Expression of biologically active proteins in a bacterial cell-free synthesis system using bacterial cells transformed to exhibit elevated levels of chaperone expression

ABSTRACT

The present disclosure describes methods and systems for improving the expression of a properly folded, biologically active protein of interest in a cell free synthesis system. The methods and systems use a bacterial cell free extract having an active oxidative phosphorylation system, and include an exogenous protein chaperone. The exogenous protein chaperone can be expressed by the bacteria used to prepare the cell free extract. The exogenous protein chaperone can be a protein disulfide isomerase and/or a peptidyl-prolyl cis-trans isomerase. The inventors discovered that the combination of a protein disulfide isomerase and a peptidyl-prolyl cis-trans isomerase produces a synergistic increase in the amount of properly folded, biologically active protein of interest.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims benefit of priority to U.S. Patent ApplicationNo. 61/813,914, filed Apr. 19, 2013, and U.S. Patent Application No.61/937,069, filed Feb. 7, 2014, the disclosure of each of which isincorporated by reference herein in its entirety.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file-58-2.TXT, created on Apr. 3, 2014,73,728 bytes, machine format IBM-PC, MS-Windows operating system, ishereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

The expression of proteins in bacterial cell free synthesis systems is awell established technique for expressing recombinant target proteins.Extracts can be made from bacteria expressing or overexpressing proteinsof interest to provide bacterial cell free synthesis systems havingaltered properties depending on the protein. However, overexpression ofproteins during bacterial growth frequently results in slower growthrates for the bacteria and lower protein synthetic activity in extractsprepared from the bacteria.

Further, expression of recombinant proteins from such extracts oftenleads to improper folding and loss of biological activity. The use ofprotein chaperones can improve the proper folding and biologicalactivity of proteins Thus, there remains a need for improved bacterialcell extracts for expressing recombinant proteins that are prepared frombacteria overexpressing chaperones where such extracts can synthesizelarge amounts of properly folded protein. These and other needs areprovided by the present invention, as set forth below.

BRIEF SUMMARY OF THE INVENTION

The present disclosure provides methods and systems for improving theexpression of biologically active and/or properly folded proteins ofinterest in a cell free synthesis system. The cell free synthesis systemcomprises a bacterial extract having an active oxidative phosphorylationsystem and the components necessary for cell free protein synthesis. Thecell free synthesis system further comprises an exogenous proteinchaperone. In some embodiments, the exogenous protein chaperone isexpressed by the bacteria used to prepare the bacterial extract.

Thus, in one aspect, a method of improving the expression levels ofbiologically active proteins in a bacterial cell free synthesis systemis described, the method comprising the steps of:

i) preparing a bacterial extract having an active oxidativephosphorylation system and comprising biologically functioning tRNA,amino acids and ribosomes necessary for cell free protein synthesis,wherein the bacteria from which the extract is prepared expresses anexogenous protein chaperone at a concentration of at least about 1gm/liter of extract;

ii) combining the bacterial extract with a nucleic acid encoding aprotein of interest to yield a bacterial cell free synthesis system;and,

iii) incubating the bacterial cell free synthesis system underconditions permitting the expression of the protein of interest to aconcentration of at least about 100 mg/L.

In a second aspect, a bacterial cell free synthesis system forexpressing biologically active proteins is described, the systemcomprising:

i) a cell free extract of bacteria having an active oxidativephosphorylation system, containing biologically functioning tRNA, aminoacids and ribosomes necessary for cell free protein synthesis andwherein an exogenous protein chaperone was expressed in the bacteria ata level of at least 1 gm/liter of extract; and,

ii) a nucleic acid encoding a protein of interest,

where said bacterial cell free synthesis system expresses a protein ofinterest to a concentration of at least about 100 mg/L.

In a third aspect, a method of expressing properly folded, biologicallyactive proteins in a bacterial cell free synthesis system is described,the method comprising the steps of:

i) preparing a bacterial extract comprising biologically functioningtRNA, amino acids, ribosomes necessary for cell free protein synthesis,a protein disulfide isomerase and a peptidyl-prolyl cis/trans isomerase,wherein the protein disulfide isomerase and the peptidyl-prolylcis/trans isomerase are present at a concentration sufficient to improvethe expression of properly folded biologically active proteins;

ii) combining the bacterial extract with a nucleic acid encoding aprotein of interest; and

iii) incubating the bacterial extract with the nucleic acid underconditions permitting the expression and proper folding of the proteinof interest.

In a fourth aspect, a bacterial cell free synthesis system forexpressing biologically active proteins is described, the systemcomprising:

i) a cell free extract of bacteria having an active oxidativephosphorylation system, containing biologically functioning tRNA, aminoacids and ribosomes necessary for cell free protein synthesis andfurther including protein disulfide isomerase and a peptidyl-prolylcis/trans isomerase,

wherein the protein disulfide isomerase and the peptidyl-prolylcis/trans isomerase are present at a concentration sufficient to improvethe expression of properly folded biologically active proteins; and

ii) a nucleic acid encoding a protein of interest,

wherein said bacterial cell free synthesis system expresses a protein ofinterest to a concentration of at least about 100 mg/L.

In a fifth aspect, a method of improving the vitality and/or growth rateof an E coli cell culture is described, the method comprising the stepsof:

i) transforming an E. coli cell with a nucleic acid expressing theprotein DsbC operably linked to a constitutive promoter; and

ii) culturing the transformed E. coli cell under conditions that permitthe overexpression of the DsbC protein to an intracellular concentrationof at least 1 mg/ml.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that eukaryotic PDI and bacterial DsbC are functionallyinterchangeable.

FIG. 2A shows a schematic illustration of the chaperone sequentialexpression screen described in the Examples.

FIG. 2B shows that IgG titer can be improved by adding bacterial cellfree system expressed protein chaperones to the bacterial cell freesynthesis system.

FIG. 3 shows that the protein chaperones Skp, SlyD and FkpA improve thesolubility and/or amount of properly assembled IgG.

FIG. 4 shows that the protein chaperone FkpA improves the solubility andfolding of IgG proteins.

FIG. 5 shows that the addition of purified FkpA to an extract containingDsbC promotes IgG folding.

FIG. 6 shows that the addition of exogenous DsbC protein added to anextract containing FkpA increases the IgG titer.

FIG. 7 shows the amount of GMCSF protein produced by the CFPS inextracts from the indicated bacterial strains that express thechaperones DsbC or FkpA.

FIG. 8 shows the growth rate of bacterial strains transformed withplasmids that express 1× or 2× copies of DsbC under the control of aconstitutive promoter (upper panel). The lower panel shows the amount ofDsbC protein present in the periplasmic lysate.

FIG. 9 shows the amount of DsbC protein produced by bacterial strainsoverexpressing 1× or 2× copies of DsbC. The upper panel shows theintracellular concentration. The lower panel shows the extractconcentration.

FIG. 10 shows the growth rate of bacterial strains transformed withplasmids that express 1× or 2× copies of FkpA under the control of aconstitutive promoter (upper panel). The lower left panel shows theamount of FkpA protein present in total extracts prepared from thebacteria expressing 1× and 2× copies of FkpA. The lower right panelshows the doubling time of the bacterial strains.

FIG. 11 shows the quantitation of FkpA concentration in extracts frombacteria expressing 1× and 2× copies of FkpA.

FIG. 12 shows the results of adding a C-terminal His tag to FkpA. (a)shows that extract levels of FkpA prepared from bacteria thatoverexpress FkpA-His (2× FkpA-His (e49)) were increased by a centrifugalspin after extract activation (pre-incubation) at 30° C. (b) shows thatextracts containing FkpA-His produced more total IgG than extractscontaining wild-type FkpA (compare 2× FkpA (e44) to 2× FkpA-His (e49)),and that the total amount of correctly assembled IgG was increased bycentrifuging the extract after activation (compare 2× FkpA final spin to2× FkpA-His final spin). Con 1 and Con 2 are control extracts preparedfrom bacteria that do not express FkpA.

FIG. 13 shows that overexpression of chaperones improves the yield ofmultiple IgGs in an Open Cell Free Synthesis system. (A) Trastuzumab,the CD30 antigen binding brentuximab, and the germline Heavy ChainsVH3-7 and VH3-23 in combination with the germline Light Chain Vk3-20were expressed in SBJY001, 2× DsbC, and 2×D+2×F extracts in the presenceof ¹⁴C-leucine and visualized by SDS-PAGE and autoradiography. (B)Assembled IgG expressed in the different extracts was quantified asdescribed in the Examples.

DEFINITIONS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULARBIOLOGY, Elsevier (4^(th) ed. 2007); Sambrook et al., MOLECULAR CLONING,A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor,N.Y. 1989); Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, JohnWiley and Sons (Hoboken, N.Y. 1995). The term “a” or “an” is intended tomean “one or more.” The term “comprise” and variations thereof such as“comprises” and “comprising,” when preceding the recitation of a step oran element, are intended to mean that the addition of further steps orelements is optional and not excluded. Any methods, devices andmaterials similar or equivalent to those described herein can be used inthe practice of this invention. The following definitions are providedto facilitate understanding of certain terms used frequently herein andare not meant to limit the scope of the present disclosure.

The term “active oxidative phosphorylation system” refers to a bacteriallysate that exhibits active oxidative phosphorylation during proteinsynthesis. For example, the bacterial lysate can generate ATP using ATPsynthase enzymes and reduction of oxygen. It will be understood thatother translation systems known in the art can also use an activeoxidative phosphorylation during protein synthesis. The activation ofoxidative phosphorylation can be demonstrated by inhibition of thepathway using specific inhibitors, such as electron transport chaininhibitors.

The term “antibody” refers to a protein functionally defined as abinding protein and structurally defined as comprising an amino acidsequence that is recognized by one of skill as being derived from theframework region of an immunoglobulin encoding gene of an animalproducing antibodies. An antibody can consist of one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to VH-CH1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab)₂ dimer into anFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Antibodies also include single chainantibodies (antibodies that exist as a single polypeptide chain), andsingle chain Fv antibodies (sFv or scFv) in which a variable heavy and avariable light chain are joined together (directly or through a peptidelinker) to form a continuous polypeptide. The single chain Fv antibodyis a covalently linked VH-VL heterodimer which may be expressed from anucleic acid including VH- and VL-encoding sequences either joineddirectly or joined by a peptide-encoding linker. Huston, et al. (1988)Proc. Nat. Acad. Sci. USA, 85: 5879-5883. While the VH and VL areconnected to each as a single polypeptide chain, the VH and VL domainsassociate non-covalently. The first functional antibody molecules to beexpressed on the surface of filamentous phage were single-chain Fv's(scFv); however, alternative expression strategies have also beensuccessful. For example Fab molecules can be displayed on phage if oneof the chains (heavy or light) is fused to g3 capsid protein and thecomplementary chain exported to the periplasm as a soluble molecule. Thetwo chains can be encoded on the same or on different replicons; theimportant point is that the two antibody chains in each Fab moleculeassemble post-translationally and the dimer is incorporated into thephage particle via linkage of one of the chains to g3p (see, e.g., U.S.Pat. No. 5,733,743). The scFv antibodies and a number of otherstructures converting the naturally aggregated, but chemically separatedlight and heavy polypeptide chains from an antibody V region into amolecule that folds into a three dimensional structure substantiallysimilar to the structure of an antigen-binding site are known to thoseof skill in the art (see, e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and4,956,778). Antibodies also includes all those that have been displayedon phage (e.g., scFv, Fv, Fab and disulfide linked Fv (Reiter et al.(1995) Protein Eng. 8: 1323-1331). Antibodies can also includediantibodies, miniantibodies and scFv-Fc fusions.

The term “bacterial derived cell free extract” refers to preparation ofin vitro reaction mixtures able to transcribe DNA into mRNA and/ortranslate mRNA into polypeptides. The mixtures include ribosomes, ATP,amino acids, and tRNAs. They may be derived directly from lysedbacteria, from purified components or combinations of both.

The term “bacterial cell free synthesis system” refers to the in vitrosynthesis of polypeptides in a reaction mix comprising biologicalextracts and/or defined reagents. The reaction mix will comprise atemplate for production of the macromolecule, e.g. DNA, mRNA, etc.;monomers for the macromolecule to be synthesized, e.g. amino acids,nucleotides, etc.; and co-factors, enzymes and other reagents that arenecessary for the synthesis, e.g. ribosomes, uncharged tRNAs, tRNAscharged with unnatural amino acids, polymerases, transcriptionalfactors, tRNA synthetases, etc.

The term “biologically active protein” refers to a protein that retainsat least some of the biological activity of the protein of interest. Thebiological activity can be determined by comparing the activity,function and/or structure of the protein of interest expressed by themethods described herein to the activity of a reference protein ofinterest. For example, if the reference protein of interest is an IgG, abiologically active protein will comprise a properly folded andassembled IgG molecule. In some embodiments, the reference protein canbe a protein expressed by a bacterial cell free synthesis system thatdoes not contain an exogenous protein chaperone. The biological activitycan also be determined using an in vitro or in vivo assay that isappropriate for the protein of interest. The biological activity of theprotein of interest can be expressed as the biological activity per unitvolume of the cell-free protein synthesis reaction mixture. In someembodiments, the biological activity of a protein produced by themethods described herein is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%,95% or 99% of the activity of a reference protein.

The term “constitutive promoter” refers to a nucleic acid sequence that,under appropriate conditions, allows for continual transcription of anucleic acid sequence or gene that is operably connected or linked tothe promoter sequence. The appropriate conditions include transcriptionfactors, such as RNA polymerase, that bind to the promoter sequence, andribonucleotides that are incorporated into the transcribed RNA.Constitutive promoters are typically unregulated promoters in that theypromote continual transcription under normal cellular conditions.

The term “disulfide isomerase” or “protein disulfide isomerase” (PDI)refers to a family of proteins comprising multiple domains, each havinga typical thioredoxin (Trx) fold. The PDI molecule has two or moreactive sites comprising a COX motif that are the sites for isomeraseactivity. In vitro, PDI catalyzes the oxidative formation, reduction, orisomerization of disulfide bonds depending on the redox potential of theenvironment. PDIs are members of a class of folding catalysts, alsocalled foldases. Folding catalysts assist folding by acceleratingcertain rate-limiting steps in the protein folding process, therebyreducing the concentration of aggregated protein folding intermediates.In addition to the isomerase function of catalyzing the formation ofdisulfide bonds, PDI also promotes the folding of polypeptides intotheir native configuration, and thus acts as a chaperone. The C-terminalregion of PDI comprises the polypeptide binding region, and is believedto be responsible for the chaperone activity. The isomerase andchaperone activities of PDI are separate and independent activities, andboth activities appear to be required for reactivation of reduced anddenatured proteins containing disulfide bonds.

In gram-negative bacteria, disulfide bond formation, reduction andisomerization are catalyzed by the Dsb (disulfide bond formation) familyof proteins, including DsbA, DsbB, DsbC, and DsbD. DsbA catalyzes theoxidative formation of disulfide bonds by transferring its active sitedisulfide to the target protein, which leaves DsbA in a reduced form.DsbB re-oxidizes DsbA, and passes its electrons to the respiratory chainto regenerate oxidized DsbB. DsbC catalyzes the rearrangement ofdisulfide bonds and is recognized as a counterpart of eukaryotic PDI.DsbC is maintained in its reduced form by DsbD. DsbC is a homodimerhaving four thiol groups is each 23 kDa subunit monomer, two in theactive site-Cys⁹⁸-Gly-Tyr-Cys¹⁰¹ (SEQ ID NO:29), and the other two aCys¹⁴¹ and Cys¹⁶³. Similar to PDI, DsbC has chaperone activity that isindependent from its isomerase activity. (See, e.g., Chen et al., J.Biol. Chem. 274:19601-19605, 1999; and Kolag, O., et al., Microbial CellFactories, 2009, 8:9). Each monomer consists of an N-terminaldimerization domain with a cystatin fold and a C-terminal catalyticdomain with a thioredoxin fold (McCarthy A. A., et al., Nat. Struct.Biol. 7:196-199, 2000). Other Dsb proteins include DsbE abd DsbG.

The term “exogenous protein chaperone” generally refers to a proteinchaperone (e.g., a recombinant protein chaperone) that is not normallyexpressed by the bacterial strain used to prepare the bacterial extract,or a recombinant protein chaperone that is expressed by a nucleic acidconstruct that is not present in the native bacterial strain. Forexample, if the native bacterial strain used to prepare the bacterialextract naturally expresses low levels of the endogenous proteinchaperone (e.g., at levels not sufficient to improve the expressionlevels of a biologically active protein of interest), the exogenousprotein chaperone can be expressed from a non-native nucleic acidconstruct, such that the nucleic acid sequences encoding the exogenousprotein chaperone are under the control of different regulatorysequences than the endogenous sequences encoding the chaperone. Forexample, the protein chaperones DsbC and FkpA are naturally occurring E.coli proteins, but their expression levels are below the limit ofdetection using the ELISA assays described herein to detect proteins inbacterial extracts. Thus, the term “exogenous” is synonymous with“heterologous,” which refers to a protein chaperone not normallyexpressed by the bacterial strain used to prepare the bacterial extract,or a nucleic acid encoding the protein chaperone that is not present inthe native bacterial strain. In some embodiments, the term refers torecombinant protein chaperones that are added to a bacterial cell freeextract, and thus are not expressed by the bacteria from which theextract was made.

The terms “identical,” “essentially identical” or percent “identity,” inthe context of two or more nucleic acids or polypeptide sequences, referto two or more sequences or subsequences that are the same or have aspecified percentage of nucleotides or amino acid residues that are thesame (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identity over a specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region, asmeasured using the BLAST and PSI-BLAST algorithms, which are describedin Altschul et al. (J. Mol. Biol. 215:403-10, 1990), and Altschul et al.(Nucleic Acids Res., 25:3389-3402, 1997), respectively. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (see the internet atncbi.nlm.nih.gov). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al. supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA89:10915-10919, 1992).

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-87, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, typically less thanabout 0.01, and more typically less than about 0.001.

When percentage of sequence identity is used in reference to apolypeptide, it is recognized that one or more residue positions thatare not otherwise identical can differ by a conservative amino acidsubstitution, in which a first amino acid residue is substituted foranother amino acid residue having similar chemical properties such as asimilar charge or hydrophobic or hydrophilic character and, therefore,does not change the functional properties of the polypeptide. Wherepolypeptide sequences differ in conservative substitutions, the percentsequence identity can be adjusted upwards to correct for theconservative nature of the substitution. Such an adjustment can be madeusing well-known methods, for example, scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions can be calculated using the algorithm described in Pearsonet al. (Meth. Mol. Biol. 24:307-331, 1994). Alignment also can beperformed by simple visual inspection and manual alignment of sequences.

The term “conservatively modified variation,” when used in reference toa particular polynucleotide sequence, refers to different polynucleotidesequences that encode identical or essentially identical (e.g., at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity over a specifiedregion) amino acid sequences, or where the polynucleotide does notencode an amino acid sequence, to essentially identical sequences.Because of the degeneracy of the genetic code, a large number offunctionally identical polynucleotides encode any given polypeptide. Forinstance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode theamino acid arginine. Thus, at every position where an arginine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleotide sequence variations are “silent variations,” which canbe considered a species of “conservatively modified variations.” Assuch, it will be recognized that each polynucleotide sequence disclosedherein as encoding a protein variant also describes every possiblesilent variation. It will also be recognized that each codon in apolynucleotide, except AUG, which is ordinarily the only codon formethionine, and UUG, which is ordinarily the only codon for tryptophan,can be modified to yield a functionally identical molecule by standardtechniques. Accordingly, each silent variation of a polynucleotide thatdoes not change the sequence of the encoded polypeptide is implicitlydescribed herein.

Furthermore, it will be recognized that individual substitutions,deletions or additions that alter, add or delete a single amino acid ora small percentage of amino acids (typically less than 10%, andgenerally less than 1%) in an encoded sequence can be consideredconservatively modified variations, provided the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative amino acid substitutions providing functionally similaramino acids are well known in the art, including the following sixgroups, each of which contains amino acids that are consideredconservative substitutes for each another:

1) Alanine (Ala, A), Serine (Ser, S), Threonine (Thr, T);

2) Aspartic acid (Asp, D), Glutamic acid (Glu, E);

3) Asparagine (Asn, N), Glutamine (Gln, Q);

4) Arginine (Arg, R), Lysine (Lys, K)

5) Isoleucine (Ile, I), Leucine (Leu, L), Methionine (Met, M), Valine(Val, V); and

6) Phenylalanine (Phe, F), Tyrosine (Tyr, Y), Tryptophan (Trp, W).

Two or more amino acid sequences or two or more nucleotide sequences areconsidered to be “substantially similar” if the amino acid sequences orthe nucleotide sequences share at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% sequence identity with each other, orwith a reference sequence over a given comparison window. Two or moreproteins are also considered substantially similar if they incorporateconservative amino acid substitutions providing functionally similaramino acids into the amino acid sequence.

The term “incubation conditions are otherwise the same” refers toexperimental conditions that, for comparison purposes, are the sameexcept that the control or reference extract does not contain or expressan exogenous protein chaperone. The term also includes a comparisonbetween a control extract that expresses or contains one class ofexogenous protein chaperone (e.g., a PDI) and an extract that expressesor contains two different classes of exogenous protein chaperones (e.g.,a PDI and a PPIase). For example, the extract can be prepared from abacterial strain that expresses or overexpresses one class of proteinchaperone (e.g., a PDI or DsbC) and a purified protein from the otherclass of protein chaperone (e.g., a purified PPIase such as FkpA) can beadded to the extract. The conditions can also include adjusting thetotal concentration of the exogenous protein chaperones (e.g., the totalconcentration of one chaperone such as PDI, or the total concentrationof the combination of two different chaperones, such as PDI and PPI) inthe bacterial extract to be the same. Otherwise, the components of thebacterial extract and the nucleic acid encoding the protein of interestare the same. Exemplary conditions that permit the expression and properfolding of a protein of interest are described in the Examples.

The terms “peptidyl prolyl isomerase,” “peptidyl prolyl cis-transisomerase” and “prolyl isomerase” (PPI or PPIase) are usedinterchangeably, and refer to a class of chaperones known as proteinfolding catalysts. PPI catalyzes the conversion of trans peptidyl prolylbonds in the amino acid proline to the cis configuration in the nativeor functional protein. PPIs can have different subunits or moduleshaving different functions, for example, a module having catalyticactivity and a module having chaperone or protein binding activity.Three families of PPIs are recognized: cyclophilins (whose isomeraseactivity is inhibited by cyclosporin A); FKBPs (FK506 binding proteins),which are inhibited by FK506 and rapamycin; and parvulins. Non-limitingexamples of cyclophilins include PpiA (RotA). Non-limiting examples ofFKBPs include FkpA, SlyD, and trigger factor (TF or tig). Non-limitingexamples of parvulins include SurA and PpiD. Additional examples of PPIsinclude CypA, PpiB, Cpr1, Cpr6, and Fpr1. FkpA, SlyD, and trigger factorare related based on sequence alignments. For FkpA, the chaperone andcatalytic activities reside in the N-terminal and C-terminal domains,respectively (Saul F. A., J. Mol. Biol. 335:595-608, 2004).

The term “deaggregase” refers to a protein chaperone that aids indeaggregating and/or solubilizing proteins of interest that areproduced, for example, in a bacterial free translation system. Suchchaperones are particularly helpful at high concentrations because theirmechanism of action is stoichiometric rather than catalytic and isbelieved to work by stabilizing hydrophobic patches of the newlysynthesized protein while the protein is folding. Examples ofdeaggregases include IbpA, IbpB, and Skp.

The term “peptide,” “protein,” and “polypeptide” are used hereininterchangeably and refer to a to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull-length proteins and truncated proteins, wherein the amino acidresidues are linked by covalent peptide bonds.

The term “properly folded protein” refers to the native conformation ofa protein or polypeptide that is biologically active or functional.Thus, the term refers to a protein or polypeptide having a tertiarystructure that in the folded state possesses a minimum of free energy.When used in reference to a recombinant protein expressed in bacteria,the term generally refers to proteins that are soluble whenoverexpressed in the cytosol, such that the properly folded recombinantprotein does not form insoluble aggregates and/or is not denatured orunfolded.

The term “synergistic” or “synergy” interchangeably refers to theinteraction of two or more agents so that their combined effect isgreater than the sum of their individual effects. Synergistic druginteractions can be determined using the median effect principle (see,Chou and Talalay (1984) Adv Enzyme Regul 22:27 and Synergism andAntagonism in Chemotherapy, Chou and Rideout, eds., 1996, Academic, pp.61-102) and quantitatively determined by combination indices using thecomputer program Calcusyn (Chou and Hayball, 1996, Biosoft, Cambridge,Mass.). See also, Reynolds and Maurer, Chapter 14 in Methods inMolecular in Medicine, vol. 110: Chemosensitivity, Vol. 1: In vitroAssays, Blumenthal, ed., 2005, Humana Press. Combination indices (CI)quantify synergy, summation and antagonism as follows: CI<1 (synergy);CI=1 (summation); CI>1 (antagonism). A CI value of 0.7-0.9 indicatesmoderate to slight synergism. A CI value of 0.3-0.7 indicates synergism.A CI value of 0.1-0.3 indicates strong synergism. A CI value of <0.1indicates very strong synergism.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The methods and systems described herein are useful for improving and/orincreasing the expression levels of biologically active proteins in acell free synthesis system, for example a bacterial cell free synthesissystem. The increased expression levels of a biologically active proteinof interest are achieved by using a bacterial extract having an activeoxidative phosphorylation system that comprises an exogenous proteinchaperone. The exogenous protein chaperone can be expressed by thebacteria used to prepare the extract. The inventors have surprisinglydiscovered that by expressing relatively large amounts of an exogenousprotein chaperone in the bacteria used to prepare the extract, increasedamounts of the biologically active protein of interest are expressed bythe cell free synthesis system. Thus, the ability of the extract toexpress large amounts of protein is surprisingly not adversely affectedby the relatively high concentration levels of the protein chaperone,such that the total amount of properly folded and biologically activeprotein produced in the cell free protein synthesis reaction issubstantially higher than the amount of properly folded and biologicallyactive protein expressed by a cell free synthesis system that does notcontain an exogenous protein chaperone. Thus, while the total amount ofthe protein of interest produced by the cell free protein synthesissystem is substantially similar to the total amount of protein producedby a cell free protein synthesis system that does not express anexogenous chaperone, the increased concentration levels of proteinchaperone in the extract results in increased amounts of properlyfolded, assembled, and biologically active protein of interest. Theinventors have also surprisingly discovered that by expressing twodifferent classes of protein chaperones (e.g., a protein disulfideisomerase and a peptidyl prolyl cis-trans isomerase), a synergisticimprovement in the expression levels of properly folded, biologicallyactive proteins is obtained. The methods and systems will now bedescribed.

To produce a biologically active protein of interest, the methods andsystems described herein use a bacterial extract having an activeoxidative phosphorylation system, and other components necessary forcell free protein synthesis, such as biologically functioning tRNA,amino acids and ribosomes. The components of the bacterial extract aredescribed in more detail below. In one aspect, the bacterial extract isprepared from a recombinant bacteria that expresses an exogenous proteinchaperone. In some embodiments, the bacteria from which the extract isprepared express the exogenous protein chaperone at a concentration ofat least about 1 gram (g)/liter (L) of extract. For example, thebacteria from which the extract is prepared can express the exogenousprotein chaperone at a concentration of at least about 1 g/liter, 2g/liter, 3 g/liter, 4 g/liter, 5 g/liter, 6 g/liter, 7 g/liter, 8g/liter, 9 g/liter, 10 g/liter or more of extract. In some embodiments,the total concentration of exogenous protein chaperone is between about1 g/L and 20 g/L, between about 1 g/L and 15 g/L, between about 1 g/Land 10 g/L, or between about 1 g/L and 5 g/L of extract. In someembodiments, the bacteria express the exogenous protein chaperone at anintracellular concentration of at least 1 mg/ml, at least 2 mg/ml, atleast 3 mg/ml, at least 4 mg/ml, at least 5 mg/ml, at least 10 mg/ml, atleast 15 mg/ml, at least 20 mg/ml, at least 30 mg/ml, or at least 40mg/ml. In some embodiments, the bacteria express the exogenous proteinchaperone at an intracellular concentration in the range of about 1mg/ml to about 40 mg/ml, about 1 mg/ml to about 20 mg/ml, about 1 mg/mlto about 15 mg/ml, about 1 mg/ml to about 10 mg/ml, or about 1 mg/ml toabout 5 mg/ml.

The exogenous protein chaperone can be any protein chaperone thatresults in increased production of properly folded and/or biologicallyfunctional proteins of interest. As described in more detail herein, theprotein chaperone can be a protein that interacts with the targetprotein of interest to assist in proper folding and/or preventaggregation of the protein of interest into nonfunctional aggregates.While not being bound by theory, molecular chaperones are thought toprevent aggregation by binding exposed hydrophobic moieties in unfolded,partially folded, or misfolded polypetides. Thus, any protein chaperonethat binds exposed hydrophobic moieties and prevents aggregation of aprotein of interest can be used in the methods described herein.

The exogenous protein chaperone can also be an enzyme that catalyzescovalent changes important for the formation of native and functionalconformations of the protein of interest. For example, in someembodiments, the exogenous protein chaperone is a protein disulfideisomerase (PDI) or a peptidyl-prolyl cis-trans isomerase (PPI). Examplesof PDI's include, but are not limited to, a mammalian PDI, a yeast PDI,or a bacterial PDI. In some embodiments, the PDI is a member of the Dsb(disulfide bond formation) family of E. coli, for example, DsbA or DsbC.In one embodiment, the exogenous protein chaperone is thioredoxin (Trx).Examples of PPI's include, but are not limited to, cyclophilins (whoseisomerase activity is inhibited by cyclosporin A); FKBPs (FK506 bindingproteins), which are inhibited by FK506 and rapamycin; and parvulins.The three families of PPIases in E. coli exhibit limited sequence andstructural similarity but share a high catalytic activity and arelatively low affinity for nonstructured peptides. As will beunderstood by those of skill in the art, the PDI and PPI chaperones canhave a modular structure that includes both a chaperone (proteinbinding) and catalytic domains. See, e.g., Kolag, O., et al., MicrobialCell Factories, 2009, 8:9; Wang, C-C., Methods in Enzymology, 2002,348:66-75. Other protein chaperones useful in the methods and systemsdescribed herein are referred to as deaggregases, including, forexample, Skp.

In another aspect, the disclosure also provides method and systems forexpressing properly folded, biologically active proteins in a bacterialcell free synthesis system using a bacterial extract comprising a PDIand a PPIase. The method comprises preparing a bacterial extractcomprising components necessary for cell free protein synthesis, such asbiologically functioning tRNA, amino acids, ribosomes. The bacterialextract further includes a protein disulfide isomerase and apeptidyl-prolyl cis-trans isomerase, wherein the protein disulfideisomerase and the peptidyl-prolyl cis-trans isomerase are present at aconcentration sufficient to improve (e.g., increase) the expression ofproperly folded biologically active proteins. In this embodiment, theexpression of a protein disulfide isomerase and a peptidyl-prolylcis-trans isomerase provides a synergistic improvement in the expressionof properly folded biologically active proteins of interest. Forexample, the expression of the protein of interest is improved to aconcentration above that concentration where one but not both of theprotein disulfide isomerase and the peptidyl-prolyl cis-trans isomeraseare present, and wherein the incubation conditions are otherwise thesame. In embodiments where the expression of a protein disulfideisomerase and a peptidyl-prolyl cis-trans isomerase provides asynergistic improvement in protein expression, the total concentrationof the protein disulfide isomerase and the peptidyl-prolyl cis-transisomerase is at least about 1 gm/liter (g/L) of extract. For example, insome embodiments, the total concentration of the protein disulfideisomerase and the peptidyl-prolyl cis-trans isomerase is at least about1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L,11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L or more of extract. In someembodiments, the total concentration of the protein disulfide isomeraseand the peptidyl-prolyl cis-trans isomerase is between about 1 g/L and20 g/L, between about 1 g/L and 15 g/L, between about 1 g/L and 14 g/L,between about 1 g/L and 10 g/L, or between about 1 g/L and 5 g/L ofextract. In some embodiments, the PDI is selected from the groupconsisting of a Dsb family protein, such as DsbA, DsbC, and DsbG, andthe PPI is selected from the group consisting of FkpA, SlyD, tig, SurA,and Cpr6.

The bacterial extracts described herein can be prepared from a bacteriathat was co-transformed with genes encoding disulfide isomerases andprolyl isomerases. The bacteria (e.g., E. coli) from which the extractis prepared can express the exogenous protein chaperone from a geneoperably linked to a constitutive promoter. In some embodiments, theexogenous protein chaperone is DsbA, DsbC, FkpA, SlyD, and/or Skp, or acombination thereof. In some embodiments, the bacterial extract is anS30 extract from E. coli.

The bacterial cell free synthesis systems described herein can have avolume between about 20 microliters and 500 liters, and the incubationtime is a time period lasting from about 1 hour to about 36 hours. Forexample, the incubation time can be between about 1 to 36 hours, about 1to 24 hours, about 1 to 18 hours, or about 1 to 12 hours.

In order to produce the protein of interest, the bacterial extract iscombined with a nucleic acid that encodes the protein of interest toyield a bacterial cell free synthesis system. The nucleic acid thatencodes the protein of interest is typically a DNA or an mRNA. Methodsfor expressing the protein of interest from a nucleic acid are describedin more detail below. The bacterial cell free synthesis system isincubated under conditions that permit the expression and/or properfolding of the protein of interest. In some embodiments, the protein ofinterest is expressed at a concentration of at least about 100 mg/L, 200mg/L, 300 mg/L, 400 mg/L, 500 mg/L, 600 mg/L, 700 mg/L, 800 mg/L, 900mg/L, or 1000 mg or more per L. Conditions for the expression of theprotein of interest are described in more detail below.

In some embodiments, the protein of interest has at least one disulfidebond in its biologically active conformation. In one embodiment, theprotein of interest has at least two proline residues. The protein ofinterest can also be an antibody or antibody fragment. In someembodiments, the protein of interest is expressed as a fusion proteinwith a chaperon protein described herein.

In another aspect, the disclosure provides a method for improving thevitality and/or growth rate of an E. coli cell culture. The methodcomprises transforming an E coli cell with a Dsb protein operably linkedto a constitutive promoter; and culturing the transformed E coli cellunder conditions that permit the overexpression of the Dsb protein. Insome embodiments, the Dsb protein is expressed at an intracellularconcentration of at least about 1 mg/ml. For example, in someembodiments, the Dsb protein is expressed at an intracellularconcentration of about 1 mg/ml to about 40 mg/ml.

In some embodiments, the protein chaperone can include a poly-amino acidtag, for example a polyhistidine (e.g., His₆; SEQ ID NO:24) tag or apoly(Ser-Arg) tag, at the N-terminus or C-terminus. In some embodiments,the poly-amino acid tag comprises charged amino acids. In someembodiments, the charged amino acids are positively charged. In someembodiments, the charged amino acids are negatively charged. In someembodiments, the poly-amino acid tag comprises polar amino acids. Insome embodiments, the poly-amino acid tag comprises alternating chargedand polar amino acids. In some embodiments, the poly-amino acid tagcomprises Ser-Arg-Ser-Arg-Ser-Arg-Ser-Arg (SEQ ID NO:25). In someembodiments, the poly-amino acid tag comprisesSer-Lys-Ser-Lys-Ser-Lys-Ser-Lys (SEQ ID NO:26). In some embodiments, thepoly-amino acid tag comprises Asp-Asp-Asp-Asp-Asp-Asp (SEQ ID NO:27). Insome embodiments, the poly-amino acid tag comprisesGlu-Glu-Glu-Glu-Glu-Glu (SEQ ID NO:28). While not being bound by anyparticular theory or mechanism of action, it is believed that theC-terminal tag increases the solubility of the chaperone, which resultsin an increase in the amount of the chaperone in extracts prepared frombacteria that express the tagged chaperone. In some embodiments, thepresence of a poly-amino acid tag resulted in an increase in the totalamount of protein of interest produced. In some embodiments,centrifuging the activated extract containing a poly-amino acid taggedchaperone increases the amount of properly assembled protein ofinterest.

General Methods

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which this invention belongs. Practitioners are particularly directedto Green, M. R. and Sambrook, J., eds., Molecular Cloning: A LaboratoryManual, 4th ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2012), and Ausubel, F. M., et al. Current Protocols inMolecular Biology (Supplement 99), John Wiley & Sons, New York (2012),which are incorporated herein by reference, for definitions and terms ofthe art. Standard methods also appear in Bindereif, Schón, & Westhof(2005) Handbook of RNA Biochemistry, Wiley-VCH, Weinheim, Germany whichdescribes detailed methods for RNA manipulation and analysis, and isincorporated herein by reference. Examples of appropriate moleculartechniques for generating recombinant nucleic acids, and instructionssufficient to direct persons of skill through many cloning exercises arefound in Green, M. R., and Sambrook, J., (Id.); Ausubel, F. M., et al.(Id.); Berger and Kimmel, Guide to Molecular Cloning Techniques, Methodsin Enzymology (Volume 152 Academic Press, Inc., San Diego, Calif. 1987);and PCR Protocols: A Guide to Methods and Applications (Academic Press,San Diego, Calif. 1990), which are incorporated by reference herein.

Methods for protein purification, chromatography, electrophoresis,centrifugation, and crystallization are described in Coligan et al.(2000) Current Protocols in Protein Science, Vol. 1, John Wiley andSons, Inc., New York. Methods for cell-free synthesis are described inSpirin & Swartz (2008) Cell-free Protein Synthesis, Wiley-VCH, Weinheim,Germany. Methods for incorporation of non-native amino acids intoproteins using cell-free synthesis are described in Shimizu et al.(2006) FEBS Journal, 273, 4133-4140.

PCR amplification methods are well known in the art and are described,for example, in Innis et al. PCR Protocols: A Guide to Methods andApplications, Academic Press Inc. San Diego, Calif., 1990. Anamplification reaction typically includes the DNA that is to beamplified, a thermostable DNA polymerase, two oligonucleotide primers,deoxynucleotide triphosphates (dNTPs), reaction buffer and magnesium.Typically a desirable number of thermal cycles is between 1 and 25.Methods for primer design and optimization of PCR conditions are wellknown in the art and can be found in standard molecular biology textssuch as Ausubel et al. Short Protocols in Molecular Biology, 5^(th)Edition, Wiley, 2002, and Innis et al. PCR Protocols, Academic Press,1990. Computer programs are useful in the design of primers with therequired specificity and optimal amplification properties (e.g., OligoVersion 5.0 (National Biosciences)). In some embodiments, the PCRprimers may additionally contain recognition sites for restrictionendonucleases, to facilitate insertion of the amplified DNA fragmentinto specific restriction enzyme sites in a vector. If restriction sitesare to be added to the 5′ end of the PCR primers, it is preferable toinclude a few (e.g., two or three) extra 5′ bases to allow moreefficient cleavage by the enzyme. In some embodiments, the PCR primersmay also contain an RNA polymerase promoter site, such as T7 or SP6, toallow for subsequent in vitro transcription. Methods for in vitrotranscription are well known to those of skill in the art (see, e.g.,Van Gelder et al. Proc. Natl. Acad. Sci. U.S.A. 87:1663-1667, 1990;Eberwine et al. Proc. Natl. Acad. Sci. U.S.A. 89:3010-3014, 1992).

When the proteins described herein are referred to by name, it isunderstood that this includes proteins with similar functions andsimilar amino acid sequences. Thus, the proteins described hereininclude the wild-type prototype protein, as well as homologs,polymorphic variations and recombinantly created muteins. For example,the name “DsbC protein” includes the wild-type prototype protein from E.coli (e.g., SEQ ID NO:1), as well as homologs from other species,polymorphic variations and recombinantly created muteins. Proteins suchas DsbC and FkpA are defined as having similar functions if they havesubstantially the same biological activity or functional capacity as thewild type protein (e.g., at least 80% of either). Proteins such as DsbCand FkpA are defined as having similar amino acid sequences if they haveat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity tothe prototype protein. The sequence identity of a protein is determinedusing the BLASTP program with the defaults wordlength of 3, anexpectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff andHenikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992).

A readily conventional test to determine if a protein homolog,polymorphic variant or recombinant mutein is inclusive of a proteinchaperone described herein is by specific binding to polyclonalantibodies generated against the prototype protein. For example, a DsbCprotein includes proteins that bind to polyclonal antibodies generatedagainst the prototype protein of SEQ ID NO:1, and an FkpA proteinincludes proteins that bind to polyclonal antibodies generated againstthe prototype protein of SEQ ID NO:6.

With regard to the reaction of a protein chaperone described herein topolyclonal antibodies, the test protein will bind under designatedimmunoassay conditions to the specified antibodies at least two timesthe background, and the specified antibodies do not substantially bindin a significant amount to other proteins present in the sample. Forexample, polyclonal antibodies raised to DsbC, encoded in SEQ ID NO:1,splice variants, or portions thereof, can be selected to obtain onlythose polyclonal antibodies that are specifically immunoreactive withDsbC and not with other proteins, except for polymorphic variants ofDsbC. This selection may be achieved by subtracting out antibodies thatcross-react with other members of the Dsb family. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, ALaboratory Manual (1988) for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity).Typically, a specific or selective reaction will be at least twicebackground signal or noise and more typically more than 10 to 100 timesbackground.

It will be understood that at least some of the chaperone proteinsdescribed herein are members of large families of related proteins withsimilar functions and various degrees of sequence homology. Thus, theprotein chaperones described herein include homologs of family membershaving similar function, for example, homologs of PDI and PPIases,homologs of Dsb proteins, homologs of FkpA proteins, etc. Thus, in someembodiments, the chaperones can have at least about 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to thechaperones described herein. Further, the data provided in the Examplesshow that eukaryotic PDI and bacterial DsbC are functionallyinterchangeable regarding their ability to produce properly assembledIgG, which provides evidence that homologs of the chaperones describedherein can be used in the methods and systems described herein.

1. Cell Free Protein Synthesis (CFPS) Technology

In order to express the biologically active proteins of interestdescribed herein, a cell free protein synthesis system can be used. Cellextracts have been developed that support the synthesis of proteins invitro from purified mRNA transcripts or from mRNA transcribed from DNAduring the in vitro synthesis reaction.

CFPS of polypeptides in a reaction mix comprises bacterial extractsand/or defined reagents. The reaction mix comprises at least ATP or anenergy source; a template for production of the macromolecule, e.g.,DNA, mRNA, etc.; amino acids, and such co-factors, enzymes and otherreagents that are necessary for polypeptide synthesis, e.g., ribosomes,tRNA, polymerases, transcriptional factors, aminoacyl synthetases,elongation factors, initiation factors, etc. In one embodiment of theinvention, the energy source is a homeostatic energy source. Alsoincluded may be enzyme(s) that catalyze the regeneration of ATP fromhigh-energy phosphate bonds, e.g., acetate kinase, creatine kinase, etc.Such enzymes may be present in the extracts used for translation, or maybe added to the reaction mix. Such synthetic reaction systems arewell-known in the art, and have been described in the literature.

The term “reaction mix” as used herein, refers to a reaction mixturecapable of catalyzing the synthesis of polypeptides from a nucleic acidtemplate. The reaction mixture comprises extracts from bacterial cells,e.g, E. coli S30 extracts. S30 extracts are well known in the art, andare described in, e.g., Lesley, S. A., et al. (1991), J. Biol. Chem.266, 2632-8. The synthesis can be performed under either aerobic oranaerobic conditions.

In some embodiments, the bacterial extract is dried. The dried bacterialextract can be reconstituted in milli-Q water (e.g., reverse osmosiswater) at 110% of the original solids as determined by measuring thepercent solids of the starting material. In one embodiment, anaccurately weighed aliquot of dried extract, representing 110% of theoriginal solids of 10 mL of extract, is added to 10 mL of Milli-Q waterin a glass beaker with a stir bar on a magnetic stirrer. The resultingmixture is stirred until the powder is dissolved. Once dissolved, thematerial is transferred to a 15 mL Falcon tube and stored at −80 Cunless used immediately.

The volume percent of extract in the reaction mix will vary, where theextract is usually at least about 10% of the total volume; more usuallyat least about 20%; and in some instances may provide for additionalbenefit when provided at at least about 50%; or at least about 60%; andusually not more than about 75% of the total volume.

The general system includes a nucleic acid template that encodes aprotein of interest. The nucleic acid template is an RNA molecule (e.g.,mRNA) or a nucleic acid that encodes an mRNA (e.g., RNA, DNA) and be inany form (e.g., linear, circular, supercoiled, single stranded, doublestranded, etc.). Nucleic acid templates guide production of the desiredprotein.

To maintain the template, cells that are used to produce the extract canbe selected for reduction, substantial reduction or elimination ofactivities of detrimental enzymes or for enzymes with modified activity.Bacterial cells with modified nuclease or phosphatase activity (e.g.,with at least one mutated phosphatase or nuclease gene or combinationsthereof) can be used for synthesis of cell extracts to increasesynthesis efficiency. For example, an E. coli strain used to make an S30extract for CFPS can be RNase E or RNase A deficient (for example, bymutation).

CFPS systems can also be engineered to guide the incorporation ofdetectably labeled amino acids, or unconventional or unnatural aminoacids, into a desired protein. The amino acids can be synthetic orderived from another biological source. Various kinds of unnatural aminoacids, including without limitation detectably labeled amino acids, canbe added to CFPS reactions and efficiently incorporated into proteinsfor specific purposes. See, for example, Albayrak, C. and Swartz, J R.,Biochem. Biophys Res. Commun., 431(2):291-5; Yang W C et al. Biotechnol.Prog. (2012), 28(2):413-20; Kuechenreuther et al. PLoS One, (2012),7(9):e45850; and Swartz J R., AIChE Journal, 58(1):5-13.

In a generic CFPS reaction, a gene encoding a protein of interest isexpressed in a transcription buffer, resulting in mRNA that istranslated into the protein of interest in a CFPS extract and atranslation buffer. The transcription buffer, cell-free extract andtranslation buffer can be added separately, or two or more of thesesolutions can be combined before their addition, or addedcontemporaneously.

To synthesize a protein of interest in vitro, a CFPS extract at somepoint comprises a mRNA molecule that encodes the protein of interest. Insome CFPS systems, mRNA is added exogenously after being purified fromnatural sources or prepared synthetically in vitro from cloned DNA usingRNA polymerases such as RNA polymerase II, SP6 RNA polymerase, T3 RNApolymerase, T7 RNA polymerase, RNA polymerase III and/or phage derivedRNA polymerases. In other systems, the mRNA is produced in vitro from atemplate DNA; both transcription and translation occur in this type ofCFPS reaction. In some embodiments, the transcription and translationsystems are coupled or comprise complementary transcription andtranslation systems, which carry out the synthesis of both RNA andprotein in the same reaction. In such in vitro transcription andtranslation systems, the CFPS extracts contain all the components(exogenous or endogenous) necessary both for transcription (to producemRNA) and for translation (to synthesize protein) in a single system.The coupled transcription and translation systems described herein aresometimes referred to as Open-Cell Free Synthesis (OCFS) systems, andare capable of achieving high titers of properly folded proteins ofinterest, e.g., high titers of antibody expression.

A cell free protein synthesis reaction mixture comprises the followingcomponents: a template nucleic acid, such as DNA, that comprises a geneof interest operably linked to at least one promoter and, optionally,one or more other regulatory sequences (e.g., a cloning or expressionvector containing the gene of interest) or a PCR fragment; an RNApolymerase that recognizes the promoter(s) to which the gene of interestis operably linked (e.g. T7 RNA polymerase) and, optionally, one or moretranscription factors directed to an optional regulatory sequence towhich the template nucleic acid is operably linked; ribonucleotidetriphosphates (rNTPs); optionally, other transcription factors andco-factors therefor; ribosomes; transfer RNA (tRNA); other or optionaltranslation factors (e.g., translation initiation, elongation andtermination factors) and co-factors therefore; one or more energysources, (e.g., ATP, GTP); optionally, one or more energy regeneratingcomponents (e.g., PEP/pyruvate kinase, AP/acetate kinase or creatinephosphate/creatine kinase); optionally factors that enhance yield and/orefficiency (e.g., nucleases, nuclease inhibitors, protein stabilizers,chaperones) and co-factors therefore; and; optionally, solubilizingagents. The reaction mix further comprises amino acids and othermaterials specifically required for protein synthesis, including salts(e.g., potassium, magnesium, ammonium, and manganese salts of aceticacid, glutamic acid, or sulfuric acids), polymeric compounds (e.g.,polyethylene glycol, dextran, diethyl aminoethyl dextran, quaternaryaminoethyl and aminoethyl dextran, etc.), cyclic AMP, inhibitors ofprotein or nucleic acid degrading enzymes, inhibitors or regulators ofprotein synthesis, oxidation/reduction adjuster (e.g., DTT, ascorbicacid, glutathione, and/or their oxides), non-denaturing surfactants(e.g., Triton X-100), buffer components, spermine, spermidine,putrescine, etc. Components of CFPS reactions are discussed in moredetail in U.S. Pat. Nos. 7,338,789 and 7,351,563, and U.S. App. Pub.Nos. 2010/0184135 and US 2010/0093024, the disclosures of each of whichis incorporated by reference in its entirety for all purposes.

Depending on the specific enzymes present in the extract, for example,one or more of the many known nuclease, polymerase or phosphataseinhibitors can be selected and advantageously used to improve synthesisefficiency.

Protein and nucleic acid synthesis typically requires an energy source.Energy is required for initiation of transcription to produce mRNA(e.g., when a DNA template is used and for initiation of translationhigh energy phosphate for example in the form of GTP is used). Eachsubsequent step of one codon by the ribosome (three nucleotides; oneamino acid) requires hydrolysis of an additional GTP to GDP. ATP is alsotypically required. For an amino acid to be polymerized during proteinsynthesis, it must first be activated. Significant quantities of energyfrom high energy phosphate bonds are thus required for protein and/ornucleic acid synthesis to proceed.

An energy source is a chemical substrate that can be enzymaticallyprocessed to provide energy to achieve desired chemical reactions.Energy sources that allow release of energy for synthesis by cleavage ofhigh-energy phosphate bonds such as those found in nucleosidetriphosphates, e.g., ATP, are commonly used. Any source convertible tohigh energy phosphate bonds is especially suitable. ATP, GTP, and othertriphosphates can normally be considered as equivalent energy sourcesfor supporting protein synthesis.

To provide energy for the synthesis reaction, the system can includeadded energy sources, such as glucose, pyruvate, phosphoenolpyruvate(PEP), carbamoyl phosphate, acetyl phosphate, creatine phosphate,phosphopyruvate, glyceraldehyde-3-phosphate, 3-Phosphoglycerate andglucose-6-phosphate, that can generate or regenerate high-energytriphosphate compounds such as ATP, GTP, other NTPs, etc.

When sufficient energy is not initially present in the synthesis system,an additional source of energy is preferably supplemented. Energysources can also be added or supplemented during the in vitro synthesisreaction.

In some embodiments, the cell-free protein synthesis reaction isperformed using the PANOx-SP system comprising NTPs, E. coli tRNA, aminoacids, Mg²⁺ acetate, Mg²⁺ glutamate, K⁺ acetate, K⁺ glutamate, folinicacid, Tris pH 8.2, DTT, pyruvate kinase, T7 RNA polymerase, disulfideisomerase, phosphoenol pyruvate (PEP), NAD, CoA, Na⁺ oxalate,putrescine, spermidine, and S30 extract.

In some embodiments, proteins containing a non-natural amino acid (nnAA)may be synthesized. In such embodiments, the reaction mix may comprisethe non-natural amino acid, a tRNA orthogonal to the 20 naturallyoccurring amino acids, and a tRNA synthetase that can link the nnAA withthe orthogonal tRNA. See, e.g., US Pat. App. Pub. No. US 2010/0093024.Alternately, the reaction mix may comprise a nnAA conjugated to a tRNAfor which the naturally occurring tRNA synthetase has been depleted.See, e.g., PCT Pub. No. WO2010/081111.

In some instances, the cell-free synthesis reaction does not require theaddition of commonly secondary energy sources, yet uses co-activation ofoxidative phosphorylation and protein synthesis. In some instances, CFPSis performed in a reaction such as the Cytomim (cytoplasm mimic) system.The Cytomim system is defined as a reaction condition performed in theabsence of polyethylene glycol with optimized magnesium concentration.This system does not accumulate phosphate, which is known to inhibitprotein synthesis.

The presence of an active oxidative phosphorylation pathway can betested using inhibitors that specifically inhibit the steps in thepathway, such as electron transport chain inhibitors. Examples ofinhibitors of the oxidative phosphorylation pathway include toxins suchas cyanide, carbon monoxide, azide, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and 2,4-dinitrophenol, antibiotics such as oligomycin,pesticides such as rotenone, and competitive inhibitors of succinatedehydrogenase such as malonate and oxaloacetate.

In some embodiments, the cell-free protein synthesis reaction isperformed using the Cytomim system comprising NTPs, E. coli tRNA, aminoacids, Mg²⁺ acetate, Mg²⁺ glutamate, K⁺ acetate, K⁺ glutamate, folinicacid, Tris pH 8.2, DTT, pyruvate kinase, T7 RNA polymerase, disulfideisomerase, sodium pyruvate, NAD, CoA, Na⁺ oxalate, putrescine,spermidine, and S30 extract. In some embodiments, the energy surbstratefor the Cytomim system is pyruvate, glutamic acid, and/or glucose. Insome embodiments of the system, the nucleoside triphosphates (NTPs) arereplaced with nucleoside monophosphates (NMPs).

The cell extract can be treated with iodoacetamide in order toinactivate enzymes that can reduce disulfide bonds and impair properprotein folding. As further described herein, the cell extract can alsobe treated with a prokaryotic disulfide bond isomerase, such as, notlimited to, E. coli DsbC and PDI. The cell extract can be treated withDsbC, FkpA and peptidyl peolyl isomerase. Glutathione disulfide (GSSG)and glutathione (GSH) can also be added to the extract at a ratio thatpromotes proper protein folding and prevents the formation of aberrantprotein disulfides.

In some embodiments, the CFPS reaction includes inverted membranevesicles to perform oxidative phosphorylation. These vesicles can beformed during the high pressure homogenization step of the preparationof cell extract process, as described herein, and remain in the extractused in the reaction mix.

The cell-free extract can be thawed to room temperature before use inthe CFPS reaction. The extract can be incubated with 50 μM iodoacetamidefor 30 minutes when synthesizing protein with disulfide bonds. In someembodiments, the CFPS reaction includes about 30% (v/v)iodoacetamide-treated extract with about 8 mM magnesium glutamate, about10 mM ammonium glutamate, about 130 mM potassium glutamate, about 35 mMsodium pyruvate, about 1.2 mM AMP, about 0.86 mM each of GMP, UMP, andCMP, about 2 mM amino acids (about 1 mM for tyrosine), about 4 mM sodiumoxalate, about 0.5 mM putrescine, about 1.5 mM spermidine, about 16.7 mMpotassium phosphate, about 100 mM T7 RNA polymerase, about 2-10 μg/mLplasmid DNA template, about 1-10 μM E. coli DsbC, and a totalconcentration of about 2 mM oxidized (GSSG) glutathione. Optionally, thecell free extract can include 1 mM of reduced (GSH).

The cell free synthesis reaction conditions may be performed as batch,continuous flow, or semi-continuous flow, as known in the art. Thereaction conditions are linearly scalable, for example, the 0.3 L scalein a 0.5 L stirred tank reactor, to the 4 L scale in a 10 L fermentor,and to the 100 L scale in a 200 L fermentor.

The development of a continuous flow in vitro protein synthesis systemby Spirin et al. (1988) Science 242:1162-1164 proved that the reactioncould be extended up to several hours. Since then, numerous groups havereproduced and improved this system (see, e.g., Kigawa et al. (1991) J.Biochem. 110:166-168; Endo et al. (1992) J. Biotechnol. 25:221-230). Kimand Choi (Biotechnol. Prog. 12: 645-649, 1996) have reported that themerits of batch and continuous flow systems can be combined by adoptinga “semicontinuous operation” using a simple dialysis membrane reactor.They were able to reproduce the extended reaction period of thecontinuous flow system while maintaining the initial rate of aconventional batch system. However, both the continuous andsemi-continuous approaches require quantities of expensive reagents,which must be increased by a significantly greater factor than theincrease in product yield.

Several improvements have been made in the conventional batch system(Kim et al. (1996) Eur. J. Biochem. 239: 881-886; Kuldlicki et al.(1992) Anal. Biochem. 206:389-393; Kawarasaki et al. (1995) Anal.Biochem. 226: 320-324). Although the semicontinuous system maintains theinitial rate of protein synthesis over extended periods, theconventional batch system still offers several advantages, e.g.convenience of operation, easy scale-up, lower reagent costs andexcellent reproducibility. Also, the batch system can be readilyconducted in multiplexed formats to express various genetic materialssimultaneously. Patnaik and Swartz (Biotechniques 24:862-868, 1998) havereported that the initial specific rate of protein synthesis could beenhanced to a level similar to that of in vivo expression throughextensive optimization of reaction conditions. It is notable that theyachieved such a high rate of protein synthesis using the conventionalcell extract prepared without any condensation steps (Nakano et al.(1996) J. Biotechnol. 46:275-282; Kim et al. (1996) Eur. J. Biochem.239:881-886). Kigawa et al. (1999) FEBS Lett 442:15-19 report highlevels of protein synthesis using condensed extracts and creatinephosphate as an energy source. These results imply that furtherimprovement of the batch system, especially in terms of the longevity ofthe protein synthesis reaction, would substantially increase theproductivity for batch in vitro protein synthesis. However, the reasonfor the early halt of protein synthesis in the conventional batch systemhas remained unclear.

The protein synthesis reactions described herein can utilize a largescale reactor, small scale, or may be multiplexed to perform a pluralityof simultaneous syntheses. Continuous reactions can use a feed mechanismto introduce a flow of reagents, and may isolate the end-product as partof the process. Batch systems are also of interest, where additionalreagents may be introduced to prolong the period of time for activesynthesis. A reactor can be run in any mode such as batch, extendedbatch, semi-batch, semi-continuous, fed-batch and continuous, and whichwill be selected in accordance with the application purpose.

2. Generating a Lysate

The methods and systems described herein use a cell lysate for in vitrotranslation of a target protein of interest. For convenience, theorganism used as a source for the lysate may be referred to as thesource organism or host cell. Host cells may be bacteria, yeast,mammalian or plant cells, or any other type of cell capable of proteinsynthesis. A lysate comprises components that are capable of translatingmessenger ribonucleic acid (mRNA) encoding a desired protein, andoptionally comprises components that are capable of transcribing DNAencoding a desired protein. Such components include, for example,DNA-directed RNA polymerase (RNA polymerase), any transcriptionactivators that are required for initiation of transcription of DNAencoding the desired protein, transfer ribonucleic acids (tRNAs),aminoacyl-tRNA synthetases, 70S ribosomes, N¹⁰-formyltetrahydrofolate,formylmethionine-tRNAf^(Met) synthetase, peptidyl transferase,initiation factors such as IF-1, IF-2, and IF-3, elongation factors suchas EF-Tu, EF-Ts, and EF-G, release factors such as RF-1, RF-2, and RF-3,and the like.

An embodiment uses a bacterial cell from which a lysate is derived. Abacterial lysate derived from any strain of bacteria can be used in themethods of the invention. The bacterial lysate can be obtained asfollows. The bacteria of choice are grown to log phase in any of anumber of growth media and under growth conditions that are well knownin the art and easily optimized by a practitioner for growth of theparticular bacteria. For example, a natural environment for synthesisutilizes cell lysates derived from bacterial cells grown in mediumcontaining glucose and phosphate, where the glucose is present at aconcentration of at least about 0.25% (weight/volume), more usually atleast about 1%; and usually not more than about 4%, more usually notmore than about 2%. An example of such media is 2YTPG medium, howeverone of skill in the art will appreciate that many culture media can beadapted for this purpose, as there are many published media suitable forthe growth of bacteria such as E. coli, using both defined and undefinedsources of nutrients. Cells that have been harvested overnight can belysed by suspending the cell pellet in a suitable cell suspensionbuffer, and disrupting the suspended cells by sonication, breaking thesuspended cells in a French press, continuous flow high pressurehomogenization, or any other method known in the art useful forefficient cell lysis The cell lysate is then centrifuged or filtered toremove large DNA fragments and cell debris.

The bacterial strain used to make the cell lysate generally has reducednuclease and/or phosphatase activity to increase cell free synthesisefficiency. For example, the bacterial strain used to make the cell freeextract can have mutations in the genes encoding the nucleases RNase Eand RNase A. The strain may also have mutations to stabilize componentsof the cell synthesis reaction such as deletions in genes such as tnaA,speA, sdaA or gshA, which prevent degradation of the amino acidstryptophan, arginine, serine and cysteine, respectively, in a cell-freesynthesis reaction. Additionally, the strain may have mutations tostabilize the protein products of cell-free synthesis such as knockoutsin the proteases ompT or lonP.

3. Proteins of Interest

The methods and systems described herein are useful for increasing theexpression of properly folded, biologically active proteins of interest.The protein of interest can be any protein that is capable of beingexpressed in a bacterial cell free synthesis system. Non-limitingexamples include proteins with disulfide bonds and proteins with atleast two proline residues. The protein of interest can be, for example,an antibody or fragment thereof, therapeutic proteins, growth factors,receptors, cytokines, enzymes, ligands, etc. Additional examples ofproteins of interest are described below.

A. Proteins with Disulfide Bonds

The methods provided herein can be used for any protein having at leastone disulfide bond in its biologically active confirmation. Disulfidebonds can stabilize tertiary protein structure by locking folding unitsinto stable conformations by linking residues in a covalent manner.

In prokaryotic cells, disulfide bonds are formed when DsbA proteindonates its disulfide bond to a newly synthesized polypeptide thatcomprises a disulfide bond in its native structure. The integralmembrane protein DsbB generates disulfide bonds within itself, which arethen transferred to DsbA. In some eukaryotic cells, the major disulfidepathway is composed of the membrane-associated flavoprotein EroI and thesoluble thioredoxin-like protein PDI. EroI, using a flavin cofactor tomediate the reoxidation of its cysteine pair by oxygen, generatesdisulfide bonds within itself, and then transfers the bonds to PDI. Inturn, PDI transfers the disulfide bonds directly to newly synthesizedpolypeptides that have not adopted their native structure.

Disulfide bonds are present in numerous proteins including, but notlimited to secreted proteins, immune proteins, extracellular matrixproteins, glycoproteins, lysosomal proteins and membrane proteins.Detailed descriptions of disulfide bonds and proteins with disulfidebonds can be found in, e.g., Fass, D. Annu. Rev. Biophys., 2012,41:63-79, Sevier, C. S. and Kaiser, C. A. Antioxidants & RedoxSignaling, 2006, 8(5):797-811 and de Marco, A., Microbial CellFactories, 2009, 8:26.

B. Proteins with Prolines

The methods provided herein can be used for any protein that has atleast two proline residues. Proline containing proteins typically favorsecondary structure elements such as turns and polyproline helices. Apolyproline helix can be an elongated, left-handed helix with torsionangles φ=−78° and ψ=+146° of the peptide backbone. A relatively highproportion of prolines can be found in proteins near the center oftransmembrane helices. Proline residues can also be found in β-turns andα-helical capping motifs, e.g., at the end of an α-helix or even one ortwo residues from the end. Prolines can also undergo cis-transisomerization which is important for proper protein folding.

Proline-rich proteins include proteins with repetitive shortproline-rich sequences, with tandemly repeated proline-rich sequences,with non-repetitive proline-rich regions, and with hydroxyproline-richproteins. Prolines residues can be found in various proteins including,but not limited to integral membrane proteins such as transporters,channels, and receptors, globular proteins, hormones, neuropeptides,mucins, immunoglobulins, and extracellular matrix proteins.

It has been shown that proline-rich peptides can enhance and/or sustainnitric oxide production in cells, potentiate argininosuccinatesynthetase activity in cells, increase intracellular concentration ofcalcium ions, and serve as ligands for SH3, WW, EVH1 or BHB domaincontaining proteins. Detailed descriptions of proline-containingproteins can be found in, e.g., Williamson, M. Biochem. J. 1994,297:249-260 and Kay et al. FASEB J., 14:231-241.

4. Chaperones

To improve the expression of a biologically active protein of interest,the present methods and systems use a bacterial extract comprising anexogenous protein chaperone. Molecular chaperones are proteins thatassist the non-covalent folding or unfolding and the assembly ordisassembly of other macromolecular structures. One major function ofchaperones is to prevent both newly synthesized polypeptide chains andassembled subunits from aggregating into nonfunctional structures. Thefirst protein chaperone identified, nucleoplasmin, assists in nucleosomeassembly from DNA and properly folded histones. Such assembly chaperonesaid in the assembly of folded subunits into oligomeric structures.Chaperones are concerned with initial protein folding as they areextruded from ribosomes, intracellular trafficking of proteins, as wellas protein degradation of misfolded or denatured proteins. Although mostnewly synthesized proteins can fold in absence of chaperones, a minoritystrictly requires them. Typically, inner portions of the chaperone arehydrophobic whereas surface structures are hydrophilic. The exactmechanism by which chaperones facilitate folding of substrate proteinsis unknown, but it is thought that by lowering the activation barrierbetween the partially folded structure and the native form, chaperonesaccelerate the desired folding steps to ensure proper folding. Further,specific chaperones unfold misfolded or aggregated proteins and rescuethe proteins by sequential unfolding and refolding back to native andbiologically active forms.

A subset of chaperones that encapsulate their folding substrates areknown as chaperonins (e.g., Group I chaperonin GroEL/GroES complex).Group II chaperonins, for example, the TRiC (TCP-1 Ring Complex, alsocalled CCT for chaperonin containing TCP-1) are thought to foldcytoskeletal proteins actin and tubulin, among other substrates.Chaperonins are characterized by a stacked double-ring structure and arefound in prokaryotes, in the cytosol of eukaryotes, and in mitochondria.

Other types of chaperones are involved in membrane transport inmitochondria and endoplasmic reticulum (ER) in eukaryotes. Bacterialtranslocation-specific chaperone maintains newly synthesized precursorpolypeptide chains in a translocation-competent (generally unfolded)state and guides them to the translocon, commonly known as atranslocator or translocation channel. A similar complex of proteins inprokaryotes and eukaryotes most commonly refers to the complex thattransports nascent polypeptides with a targeting signal sequence intothe interior (cisternal or lumenal) space of the endoplasmic reticulum(ER) from the cytosol, but is also used to integrate nascent proteinsinto the membrane itself (membrane proteins). In the endoplasmicreticulum (ER) there are general chaperones (BiP, GRP94, GRP170), lectin(calnexin and calreticulin) and non-classical molecular chaperones(HSP47 and ERp29) helping to fold proteins. Folding chaperone proteinsinclude protein disulfide isomerases (PDI, DsbA, DsbC) and peptidylprolyl cis-trans isomerases (PPI, FkpA, SlyD, TF).

Many chaperones are also classified as heat shock proteins (Hsp) becausethey are highly upregulated during cellular stress such as heat shock,and the tendency to aggregate increases as proteins are denatured byelevated temperatures or other cellular stresses. Ubiquitin, which marksproteins for degradation, also has features of a heat shock protein.Some highly specific ‘steric chaperones’ convey unique structuralconformation (steric) information onto proteins, which cannot be foldedspontaneously. Other functions for chaperones include assistance inprotein degradation, bacterial adhesin activity, and response to priondiseases linked to protein aggregation.

Enzymes known as foldases catalyze covalent changes essential for theformation of the native and functional conformations of synthesizedproteins. Examples of foldases include protein disulfide isomerase(PDI), which acts to catalyze the formation of native disulfide bonds,and peptidyl prolyl cis-trans isomerase (PPI), which acts to catalyzeisomerization of stable trans peptidyl prolyl bonds to the cisconfiguration necessary for the functional fold of proteins. Theformation of native disulfides and the cis-trans isomerization of prolylimide bonds are both covalent reactions and are frequently rate-limitingsteps in the protein folding process. Recently proposed to be chaperoneproteins, in stoichiometric concentrations foldases increase thereactivation yield of some denatured proteins. Other examples ofchaperone proteins include deaggregases such as Skp, and the redoxproteins Trr1 and Glr1.

In some embodiments, the protein chaperone can be co-expressed withanother protein(s) that functions to increase the activity of thedesired protein chaperone. For example, the Dsb proteins DsbA and DsbCcan be coexpressed with DsbB and DsbD, which oxidize and reduce DsbA andDsbC, respectively.

5. Transforming Bacteria with Genes Encoding the Chaperones

The bacterial extracts used in the methods and systems described hereincontain an exogenous protein chaperone. The exogenous protein chaperonesdescribed herein can be added to the extract, or can be expressed by thebacteria used to prepare the cell free extract. In the latterembodiment, the exogenous protein chaperone can be expressed from a geneencoding the exogenous protein chaperone that is operably linked to apromoter that initiates transcription of the gene.

Promoters that may be used in the present invention include bothconstitutive promoters and regulated (inducible) promoters. Thepromoters may be prokaryotic or eukaryotic depending on the host. Amongthe prokaryotic (including bacteriophage) promoters useful for practiceof this invention are lac, T3, T7, lambda Pr′P1′ and trp promoters.Among the eukaryotic (including viral) promoters useful for practice ofthis invention are ubiquitous promoters (e.g. HPRT, vimentin, actin,tubulin), intermediate filament promoters (e.g. desmin, neurofilaments,keratin, GFAP), therapeutic gene promoters (e.g. MDR type, CFTR, factorVIII), tissue-specific promoters (e.g. actin promoter in smooth musclecells), promoters which respond to a stimulus (e.g. steroid hormonereceptor, retinoic acid receptor), tetracycline-regulatedtranscriptional modulators, cytomegalovirus immediate-early, retroviralLTR, metallothionein, SV-40, E1a, and MLP promoters.Tetracycline-regulated transcriptional modulators and CMV promoters aredescribed in WO 96/01313, U.S. Pat. Nos. 5,168,062 and 5,385,839, theentire disclosures of which are incorporated herein by reference.

In some embodiments, the promoter is a constitutive promoter. Examplesof constitutive promoters in bacteria include the spc ribosomal proteinoperon promotor P_(spc), the β-lactamase gene promotor P_(bla) ofplasmid pBR322, the P_(L) promoter of phage λ, the replication controlpromoters P_(RNAI) and P_(RNAII) of plasmid pBR322, the P1 and P2promoters of the rrnB ribosomal RNA operon, the tet promoter, and thepACYC promoter.

6. Quantitatively Measuring Protein of Interest and Chaperones

The quantity of the protein of interest produced by the methods andsystems described herein can be determined using any method known in theart. For example, the expressed protein of interest can be purified andquantified using gel electrophoresis (e.g., PAGE), Western analysis orcapillary electrophoresis (e.g., Caliper LabChip). Protein synthesis incell-free translation reactions may be monitored by the incorporation ofradiolabeled amino acids, typically, ³⁵ S-labeled methionine or¹⁴C-labeled leucine. Radiolabeled proteins can be visualized formolecular size and quantitated by autoradiography after electrophoresisor isolated by immunoprecipitation. The incorporation of recombinant Histags affords another means of purification by Ni²⁺ affinity columnchromatography. Protein production from expression systems can bemeasured as soluble protein yield or by using an assay of enzymatic orbinding activity.

The amount of chaperone protein that is added to the cell free synthesissystem can be quantified by including a radioactive amino acid, such as¹⁴C-Leucine, in the bacterial cell culture used to prepare the bacterialextract, and quantifying the amount of expressed protein chaperone by,for example, precipitating the radioactive protein using trichloroaceticacid (TCA), and measuring the total amount of radioactivity recovered.The amount of chaperone can also be measured immunologically, forexample, by an ELISA in which monoclonal or polyclonal antibodiesagainst the chaperone are used to detect and quantify chaperone proteinimmobilized in plates or on a Western blot.

7. Quantitatively Measuring Biological Activity and Proper Folding ofExpressed Proteins

The biological activity of a protein of interest produced by the methodsdescribed herein can be quantified using an in vitro or in vivo assayspecific for the protein of interest. The biological activity of theprotein of interest can be expressed as the biological activity per unitvolume of the cell-free protein synthesis reaction mixture. The properfolding of an expressed protein of interest can be quantified bycomparing the amount of total protein produced to the amount of solubleprotein. For example, the total amount of protein and the solublefraction of that protein produced can be determined by radioactivelylabeling the protein of interest with a radiolabeled amino acid such as¹⁴C-leucine, and precipitating the labeled proteins with TCA. The amountof folded and assembled protein can be determined by gel electrophoresis(PAGE) under reducing and non-reducing conditions to measure thefraction of soluble proteins that are migrating at the correct molecularweight. Under non-reducing conditions, protein aggregates can be trappedabove the gel matrix or can migrate as higher molecular weight smearsthat are difficult to characterize as discrete entities, whereas underreducing conditions and upon heating of the sample, proteins containingdisulfide bonds are denatured, aggregates are dissociated, and expressedproteins migrate as single bands. Methods for determining the amount ofproperly folded and assembled antibody proteins are described in theExamples. Functional activity of antibody molecules can be determinedusing an immunoassay, for example, an ELISA.

EXAMPLES Example 1

This example demonstrates that chaperone proteins expressed by abacterial cell free protein synthesis system increase the amount ofproperly assembled IgG expressed by the cell free protein synthesissystem, and that the combination of a bacterial PDI and a PPI actedsynergistically to increase the amount of properly assembled IgG.

Engineering of a bacterial endoplasmic reticulum for the rapidexpression of immunoglobulin proteins.

Materials and Methods:

Small-scale cell-free expression. 100 μl cell-free protein synthesisreactions were run at 30° C. for 12 hr in a 96-well microtiter plate at650 rpm in a VWR Thermomixer in the presence of 10 μg/mL DNA (2.5 μg/mLtrastuzumab light chain DNA, 7.5 μg/mL trastuzumab heavy chain DNA inthe expression vector pYD317). Cell-free extracts were treated with 50μM iodoacetamide for 30 min at RT (20° C.) and added to a premix ofcomponents. The final concentration in the protein synthesis reactionwas 30% cell extract (v/v), 2 mM GSSG, 8 mM magnesium glutamate, 10 mMammonium glutamate, 130 mM potassium glutamate, 35 mM sodium pyruvate,1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mM amino acids (except1 mM for tyrosine and phenylalanine), 4 mM sodium oxalate, 1 mMputrescine, 1.5 mM spermidine, 15 mM potassium phosphate, 20 ug/mL T7RNAP, unless otherwise indicated.

Interchangeability of PDI and DsbC. Cell-free protein synthesisreactions were run at varying concentrations of PDI and DsbC tounderstand the requirements for disulfide bond isomerases on IgG foldingand assembly. 0-5 uM recombinant PDI was added to cell-free reactions incombination with 0-13 uM recombinant DsbC. 100 μl cell-free reactionswere run with 30% control extract for 12 hr at 30° C. in a 96-wellmicrotiter plate at 650 rpm in a VWR Thermomixer in the presence of 8μg/mL HC-HIS6 DNA and 2 μg/mL LC DNA. The reactions were subsequentlycentrifuged at 5000×g for 10 minutes and supernatants were diluted2-fold with PBS prior to purification on IMAC Phytips (200 μl tips, 5 μlresin bed) using a Biomek robotic system. Samples were eluted in 20 mMTris pH8, 300 mM NaCl, 500 mM imidazole and the eluted IgG wasquantified using capillary electrophoresis on a Caliper LapChip GXII.

Chaperone sequential expression screen. Candidate chaperones were clonedinto the cell-free expression plasmid pYD317. From these plasmids, PCRfragments were generated that contained the chaperone gene sandwichedbetween T7 promoter and terminator sequences. Chaperones weresubsequently expressed from these PCR fragments by cell-free proteinsynthesis under standard microtiter plate conditions for 16 hr at 30° C.To stabilize the PCR fragments against DNA degradation, 40 ug/mL GamSprotein was added to the reactions. Chaperone-expressing extract wassubsequently centrifuged at 5000×g for 10 minutes andchaperone-containing supernatants were added into new cell-freereactions at 20% (v/v) for the expression of IgG (8 μg/mL trastuzumabheavy chain DNA and 2 μg/mL trastuzumab light chain DNA) in the presenceof ¹⁴C-leucine. IgG titers were calculated based on the rate ofincorporation of ¹⁴C-leucine into the IgG molecule, as previouslydescribed (MAbs. 2012 Mar. 1; 4(2)). Chaperone-related improvements inIgG titer were expressed as a fold improvement over the addition of aGFP-expressing extract. To estimate the amount of chaperone being addedto the IgG expression reactions, chaperone cell-free reactions were alsorun in the presence of ¹⁴C-leucine and the expressed protein wasquantified.

2× DsbC and 2× FkpA extracts. Bicistronic plasmids of the bacterialgenes DsbC (2× DsbC) and FkpA (2× FkpA) behind a constitutive promoter(pACYC) were generated and transformed into bacteria. These strains weregrown to log phase and lysed for the production of cell-free extract, asdescribed in Yang W. C. et al. Biotechnol. Prog. (2012), 28(2):413-20.FkpA protein was added to an IgG cell-free reaction using 2× DsbCextract to test if FkpA would further improve IgG folding and assembly.The reverse experiment was performed by the addition of 13 μM DsbCprotein to a cell-free reaction with 2× FkpA extract.

Results:

Interchangeability of PDI and DsbC. To better understand the dependenceof IgG folding and assembly on eukaryotic and bacterial disulfide bondisomerases, IgG cell-free protein synthesis reactions were run atvarying concentrations of PDI and DsbC. IgG was expressed in cell-freereactions in the presence of 0-5 μM PDI in combination with 0-13 μMDsbC. Expressed IgG-His was purified by N⁺⁺ resin and quantified bycapillary electrophoresis (FIG. 1). In the absence of DsbC, IgG washighly dependent on PDI for folding (FIG. 1, closed circles). However,as the concentration of DsbC in the reaction increased, the dependenceon PDI fell such that at 6.4 μM DsbC, there was no additional benefitattributable to PDI in the reaction (FIG. 1, open triangles).Furthermore, by increasing the concentration of DsbC in the reaction, wesaw marked improvements in IgG titers beyond what we had previouslyobserved (FIG. 1, open circles). In effect, we observed the efficientsubstitution of a eukaryotic disulfide bond isomerase with a bacterialchaperone of a similar function in the folding of a eukaryotic protein.

Chaperone sequential expression screen. In vivo, eukaryotic chaperonesare known to play an important role in the folding and assembly of IgG.Therefore, expression of IgG molecules in bacterial systems which lackthese physiological foldases has been challenging (REFS). As such, weundertook a screening approach to identify chaperone proteins that wouldbe positive effectors of IgG folding and/or assembly. Candidatechaperones were expressed in our cell-free system and expressedchaperones were subsequently added into new cell-free reactions for theexpression of IgG. Any improvements in IgG folding were expressed as animprovement in titer over the addition of a GFP-expressing controlextract, a protein unlikely to interact with IgG. In order to improvethe throughput of the screen, chaperones were not purified from theextract before being added to IgG reactions. Because of this, we wantedto ensure that chaperone DNA was not being transcribed and expressed insubsequent IgG reactions. As such, chaperone proteins were expressedfrom PCR template which is significantly more labile than plasmid DNA.The addition of GamS protein helped preserve the PCR template, such thatsufficient levels of chaperone protein could be synthesized.

Several families of chaperones were of particular interest given theirrole in folding IgG in vivo. PPIases, foldases, deaggregases, and redoxproteins from bacterial, yeast, and human species were tested. Among theredox chaperones, we found that PDI (yeast homologue) and DsbCsignificantly aided IgG formation, consistent with our previous findings(FIG. 2B). Interestingly, human PDI (hPDI) did not significantly impactIgG folding, probably due to its poor expression in cell extract whichdid not allow it to be added in sufficient quantities to aid IgGfolding. By contrast, the bacterial protein DsbC expressed very well inthe extract, allowing the addition of ˜5 uM DsbC to the IgG reaction(DsbC was expressed at ˜25 uM and it was added at 20% to an IgGreaction). Among the PPIases tested, several proved to be beneficial toIgG expression (FIG. 2B). From these, we decided to follow-up on Skp,SlyD, and FkpA.

Purified Skp, SlyD, and FkpA can improve IgG titers. To confirm our hitsfrom the chaperone screen, we expressed and purified Skp, SlyD, and FkpAand added them back into IgG cell-free protein synthesis reactions (FIG.3). For the chaperone Skp, we saw that Skp aided the solubility of HCand LC, but did not increase the amount of assembled IgG significantly.However, for the prolyl isomerases, SlyD and FkpA, we observed that themore of these chaperones we added, the amount of soluble proteins andassembled IgG increased proportionately. We reasoned that prolylisomerization was a function that was previously limiting for IgGformation in our cell-free protein synthesis system and the addition ofthese exogenous proteins improved IgG folding and assembly dramatically.Because of the vast improvements observed with DsbC and FkpA, we decidedto further characterize their roles in IgG folding.

FkpA and DsbC work synergistically to fold and assemble IgG. To betterunderstand the roles that FkpA and DsbC play in IgG formation, weindependently evaluated their contributions to IgG folding (FIG. 4).Interestingly, the addition of FkpA significantly reduced the degree ofhigher molecular weight aggregates formed during HC and LC synthesis.With increasing amounts of FkpA, we also observe the formation of IgG,as well as a number of partially assembled products. These proteinsmigrated as fuzzy bands, suggesting that they may represent mixedpopulations of cross-disulfide bonded proteins. The addition of DsbC, onthe other hand, generated clear sharp bands of IgG. However, withoutFkpA, a significant proportion of the expressed proteins formed higherorder aggregates that could not completely enter the SDS-PAGE gel.

When the two chaperones were combined into the same IgG reaction, theyacted synergistically to fold IgG (FIG. 5). HC and LC were expressed ina DsbC-containing extract (2× DsbC) and different amounts of exogenousFkpA protein were added. At 50 uM FkpA, on the order of 900 ug/mL ofassembled IgG could be expressed. To follow-up on this, a bacterialstrain overexpressing FkpA was engineered from which cell extract wasgenerated. IgG was synthesized from FkpA extract with the addition ofexogenous DsbC protein (FIG. 6). IgG was produced at ˜600 ug/mL withreduced aggregation under our standard conditions of 30% extract (v/v).To further increase the concentration of FkpA in each reaction, wetitrated up the FkpA-containing extract in the reaction which broughtthe IgG titers to >900 ug/mL (FIG. 6).

The above example demonstrates that the combination of two differentclasses of protein chaperones, a PDI and a PPI, provides a synergisticeffect on proper protein folding and assembly in a cell free expressionsystem.

Example 2

This example demonstrates that overexpression of exogenous proteinchaperones in bacterial strains used to prepare cell extracts does notinhibit the production of a protein of interest such as GMCSF.

Strain Descriptions:

SBDG028: SBJY001+pACYC 2× DsbC+ΔRF1

SBDG031: SBJY001+pACYC 2× DsbC

SBDG044: SBJY001+pACYC 2× FkpA

SBDG049: SBJY001+pACYC 2× FkpA-6×His

Cell Extract Preparation:

Extracts from E. coli strains SBDG028, SBDG031, SBDG044 and SBDG049 wereprepared essentially as described in Zawada et al., Biotechnology andBioengineering Vol. 108, No. 7, July 2011.

GMCSF CFPS Reaction

The cell-free reaction procedure for GMCSF protein production wasperformed as described in Zawada et al. Biotechnology and BioengineeringVol. 108, No. 7, July 2011, which is incorporated by reference herein inits entirety.

FIG. 7 shows the amount of GMCSF protein produced by the CFPS inextracts from the indicated strains that overexpress DsbC or FkpA. Incontrol extracts prepared from bacteria that do not express an exogenousDsbC or FkpA, very little GMCSF is produced (data not shown).

Example 3

This example demonstrates that bacterial cells overexpressing proteinchaperones have similar growth rates as bacteria that do not overexpressprotein chaperones.

Methods: Bacterial strains were transformed with recombinant plasmidsthat express one (1×) or two (2×) copies DsbC and FkpA, as described inExample 1. These strains were grown to log phase lysed for theproduction of cell-free extract. The growth rates (doubling times) forthe strains were determined, and the amount of protein chaperoneproduced by the bacteria strains was quantified using Western analysisand/or ELISA.

To determine the intracellular concentration of the expressed proteinchaperones, the periplasm of shake flask grown cells was lysed usingosmotic shock. The periplasmic lysate was separated by gelelectrophoresis with standards of known DsbC concentration. Densitometrywas used to compare the intensity of the standard DsbC bands to theintensity of the bands in the periplasmic lysate. The intensity of thebands was used to determine the DsbC concentration in the lysate, whichwas used to back calculate the concentration of DsbC in the cells.

The amount of chaperone protein in the cell-free extracts was determinedby ELISA. The ELISA to determine DsbC and FkpA titers in cell-freeextract is the Direct ELISA format. The assay consists of coating anassay plate with standards and samples, then allowing an antibody thatrecognizes DsbC or FkpA to bind, washing away excess DsbC and FkpAantibody, introducing an HRP conjugated secondary antibody to rabbit IgG(the DsbC and FkpA antibodies were produced in rabbit), washing awayexcess conjugated secondary antibody, and then using an ABTS substrateto detect the HRP present on the conjugated secondary antibody. PurifiedDsbC and FkpA with known concentrations were used to create a 7 pointstandard curve to use in the determination of sample concentrations.

DsbC: MSD (Minimum Sample Dilution): 1/120,000; LLOQ (Lower Limit ofQuantitation) at MSD: 187.5 ug/ml.

FkpA: MSD (Minimum Sample Dilution): 1/75,000; LLOQ (Lower Limit ofQuantitation) at MSD: 390 ug/ml

Results:

FIG. 8 shows the growth rate of bacterial strains transformed withplasmids that express 1× or 2× copies of DsbC under the control of aconstitutive promoter. The growth rates of strains expressing 1× and 2×copies of DsbC were similar to a control strain that was not transformedwith the expression plasmids. The lower panel of FIG. 8 shows the amountof DsbC protein present in the periplasmic lysate, as described above.

FIG. 9 shows the amount of DsbC protein produced by the bacterialstrains overexpressing 1× or 2× copies of DsbC. The upper panel showsthe intracellular concentration, determined as described above. Thelower panel shows the extract concentration, determined by ELISA.

FIG. 10 shows growth rate of bacterial strains transformed with plasmidsthat express 1× or 2× copies of FkpA under the control of a constitutivepromoter. The growth rates of strains expressing 1× and 2× copies ofFkpA were similar to a control strain that was not transformed with theexpression plasmids. The lower left panel of FIG. 10 shows the amount ofFkpA protein present in total extracts prepared from the bacteriaexpressing 1× and 2× copies of FkpA. FIG. 11 shows the quantitation ofFkpA concentration in extracts from bacteria expressing 1× and 2× copiesof FkpA.

The results of representative ELISA experiments are shown in the Tablesbelow. The ELISA data for FkpA is from a different extract preparationthan that shown in FIG. 9, which accounts for the different DsbCconcentrations.

TABLE 1 DsbC concentrations determined in extracts by ELISA. DsbCStandard Titer Deviation Strain Description (mg/ml) (mg/ml) SBJY- WTControl Extract <0.188 N/A 001 SBDG- 1x DsbC Extract 1.084 0.016 026SBDG- 2x DsbC Extract 3.155 0.351 028 SBDG- 2x DsbC Extract 3.267 0.353031 (No RF1 Deletion) SBDG- 3x DsbC Extract 2.854 0.272 033

TABLE 2 FkpA concentrations determined in extracts by ELISA. FkpAStandard Titer Deviation Strain Description (mg/ml) (mg/ml) SBJY- WTControl Extract <0.390 N/A 001 SBDG- 1x FkpA 3.029 0.305 034 SDDG- 2xFkpA 5.121 0.076 044 SBDG- 2x FkpA with leader sequences 1.960 0.252 048removed for cytoplasmic expression SBDG- 2x FkpA w/6xHis (SEQ ID NO: 24)5.415 0.147 049 tag SBDG- 1x FkpA Pc7 Promoter 2.786 0.059 052

This example demonstrates that recombinant bacterial strains thatoverexpress chaperone proteins are capable of rapid growth and areuseful for preparing high quality extracts for cell free proteinsynthesis.

Example 4

This example shows that including a poly-charged amino acid tag on theC-terminal of the chaperone FkpA increased the amount of FkpA in theextract, and increased the amount of total protein produced by the cellfree protein synthesis system.

The gene encoding FkpA was cloned with either a His₆ (SEQ ID NO:24) or(Ser-Arg)₄ (SEQ ID NO:25) tag on the C-terminus in vector pACYC-Pc.These vectors were transformed into strain SBJY001 and extract wasproduced as described above. An FkpA ELISA showed that extract levels ofthe His-tagged FkpA variants were increased by a final centrifugal spinof the extract, post-activation (FIG. 12 a). Compared to extractcontaining wt FkpA, extracts containing these solubility-tagged FkpAproteins produced more total protein. In addition, assembled IgG levelswere enhanced by a final spin of the extract after activation (FIG. 12b).

This example demonstrates that adding a poly-charged amino acid tag onthe C-terminus of FkpA increased the amount of FkpA expressed bybacteria used to make the extract and increased the amount of totalprotein produced. Further, for extracts containing the C-terminalHis-tagged FkpA, spinning the extract down after activation resulted inan increase in the amount of correctly assembled IgG.

Example 5

This example demonstrates that genomic integration of the chaperonesdsbC and FkpA in two independent bacterial strains resulted in cellswith a high growth rate that produced high chaperone levels, andcell-free extracts derived from these strains contained high levels ofboth chaperones and supported cell-free synthesis of high levelsrecombinant IgG and GMC-SF.

Strain 108

Strain SBDG108 is a derivative of SBMT095. This strain has 2 copies ofdsbC integrated onto the chromosome into the galK locus behind a mediumstrength constitutive promoter prepared using homologous recombination.SBMT095 was made competent and then transformed with pACYC-Pc0-2× FkpA,a medium copy plasmid with two copies of FkpA behind a constitutivepromoter. Both copies coded for wild type E. coli FkpA, but one gene hadbeen synthesized to reduce nucleotide homology to the WT gene, enablingeach to be propagated stably in the same plasmid.

In a standard extract fermentation using DM80-80 in batch mode, strainSBDG108 was capable of achieving a high growth rate while stillproducing very high chaperone levels (See Table 3).

TABLE 3 Properties of 108 in extract fermentation. Intracellular DsbCtiter  4.1 mg/ml Intracellular FkpA titer 13.9 mg/ml Specific GrowthRate 0.49/h

The extract made from strain 108 contained high levels of bothchaperones and supported cell-free synthesis of very high levels ofrecombinant IgGs and other proteins (see Table 4).

TABLE 4 Cell-Free protein titers. GMC-SF 0.44 mg/ml Trastuzumab  1.1mg/ml

Strain 150

Strain SBMT150 is a derivative of SBHS016, a KGK10 derivative with ompTsensitive RF1. To produce SBMT150, 2 copies of DsbC were integrated ontothe chromosome into the xy1A locus. Two copies of FkpA were integratedinto the galK locus. Both chromosomal integrations were introduced withhomologous recombination.

In a standard extract fermentation using DM80-80 in batch mode, strainSBMT150 was capable of achieving a high growth rate while stillproducing high chaperone levels (see Table 5). Because the chaperonesare overexpressed from the genome, no antibiotics are required duringthe fermentation of this strain.

TABLE 5 Properties of 150 in extract fermentation. Intracellular DsbCtiter 2.5 mg/ml Intracellular FkpA titer 3.4 mg/ml Specific Growth Rate.071/h

The extract made from strain 108 contained high levels of bothchaperones and supported cell-free synthesis of high levels ofrecombinant IgGs and other proteins, as shown in the Table 6 below.

TABLE 6 Cell Free Protein Titers. GMC-SF 0.46 mg/ml Trastuzumab 0.49mg/ml

In summary, this example demonstrates that bacterial strains can beengineered to stably incorporate chaperone expression cassettes thatexpress high levels of chaperone proteins without compromising growthrates, and that cell free extracts derived from these strains yield highlevels of recombinant proteins of interest.

Example 6

This example shows that extracts derived from bacterial cells thatoverexpress the DsbC and FkpA chaperones can improve the expression andassembly of multiple different IgG's.

Methods:

2× DsbC and 2× FkpA Extracts.

The E. coli strain SBJY001 (Yin G, et al., Aglycosylated antibodies andantibody fragments produced in a scalable in vitrotranscription-translation system. mAbs 2012; 4) was transformed withpACYC-based chaperone overexpression plasmids and harvested in log phaseto make cellular extracts. Plasmids carrying one copy (1× DsbC) or twotandem copies (2× DsbC) of dsbC behind the E. coli promoter Mt-cons-10(Thouvenot B. et al. The strong efficiency of the Escherichia coli gapAP1 promoter depends on a complex combination of functional determinants.Biochem J2004; 383:371-82) were generated and transformed into bacteria,as were one copy (1× FkpA) or two copies (2× FkpA) of fkpA. Thesestrains were grown to log phase and lysed for the production ofcell-free extract, as described (Zawada J. F. et al. Microscale tomanufacturing scale-up of cell-free cytokine production—a new approachfor shortening protein production development timelines. BiotechnolBioeng 2011; 108:1570-8). The IgG-producing activities of each of theseextracts were tested, either alone or in combination with exogenouslyadded purified protein. A bacterial strain SBHS016 (derived frombacterial strain SBJY001) optimized for OCFS extracts was furthermodified to enhance the production of DsbC protein. This strain has dualtandem copies of dsbC integrated into the bacterial galK locus,constitutively expressed using a modified MT-cons-10 promoter (ThouvenotB. et al. Biochem J 2004; 383:371-82). This is in addition to the wildtype gene at the normal dsbC locus. The dual tandem gene cassettecontains one copy of the parental dsbC gene, and one copy of a syntheticversion of the dsbC gene designed to encode the wild type protein, butwith altered codons to suppress unwanted sequence recombination withother versions of dsbC gene elsewhere in the genome. This DsbCoverexpressing strain was transformed with the 2× FkpA plasmid toproduce strain ‘2×D+2×F’.

Results:

A panel of different IgG's were translated in a bacterial in vitrotranscription/translation system described herein. The IgG's weretranslated in a control extract (SBJY001), a DsbC extract (2× DsbCextract), and a DsbC+FkpA extract (2×D+2×F). The panel included thetherapeutic antibodies trastuzumab (an anti-Her2 IgG1) and brentuximab(an anti-CD30 IgG1), in addition to two germline Heavy Chains VH3-7 andVH3-23 in combination with the Light Chain Vk3-20. As shown in FIG. 13,expression of the IgG's in the 2× DsbC extract dramatically improved theyield of all four IgG's. Further improvements were observed in theDsbC+FkpA extract, bringing expression levels to 1 g/L for bothtrastuzumab and brentuximab and nearly 1.5 g/L for the germline IgGs.

This example demonstrates that extracts from engineered bacteria thatoverexpress the chaperones DsbC and FkpA can increase the expression ofa wide-range of immunoglobulin proteins in a OCFS coupledtranscription-translation system.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, sequence accessionnumbers, patents, and patent applications cited herein are herebyincorporated by reference in their entirety for all purposes.

Informal Sequence Listing:SEQ ID NO: 1 NP_417369 protein disulfide isomerase II [Escherichia coli str. K-12 substr. MG1655] (DsbC; xprA) (UniProt P0AEG6)   1 MKKGFMLFTL LAAFSGFAQA DDAAIQQTLA KMGIKSSDIQ PAPVAGMKTV LTNSGVLYIT  61DDGKHIIQGP MYDVSGTAPV NVTNKMLLKQ LNALEKEMIV YKAPQEKHVI TVFTDITCGY 121CHKLHEQMAD YNALGITVRY LAFPRQGLDS DAEKEMKAIW CAKDKNKAFD DVMAGKSVAP 181ASCDVDIADH YALGVQLGVS GTPAVVLSNG TLVPGYQPPK EMKEFLDEHQ KMTSGKSEQ ID NO: 2 NP_418297 periplasmic protein disulfide isomerase I [Escherichia coli str. K-12 substr. MG1655](DsbA; dsf; ppfA) (UniProt P0AEG4)   1MKKIWLALAG LVLAFSASAA QYEDGKQYTT LEKPVAGAPQ VLEFFSFFCP HCYQFEEVLH  61 ISDNVKKKLP EGVKMTKYHV NFMGGDLGKD LTQAWAVAMA LGVEDKVTVP LFEGVQKTQT 121IRSASDIRDV FINAGIKGEE YDAAWNSFVV KSLVAQQEKA AADVQLRGVP AMFVNGKYQL 181NPQGMDTSNM DVFVQQYADT VKYLSEKKSEQ ID NO: 3 NP_415703 oxidoreductase that catalyzes reoxidation of DsbAprotein disulfide isomerase I [Escherichia coli str. K-12 substr. MG1655](DsbB; roxB; ycgA) (UniProt P0A6M2)   1MLRFLNQCSQ GRGAWLLMAF TALALELTAL WFQHVMLLKP CVLCIYERCA LFGVLGAALI  61 GAIAPKTPLR YVAMVIWLYS AFRGVQLTYE HTMLQLYPSP FATCDFMVRF PEWLPLDKWV 121PQVFVASGDC AERQWDFLGL EMPQWLLGIF IAYLIVAVLV VISQPFKAKK RDLFGRSEQ ID NO: 4 NP_418559 fused thiol:disulfide interchange protein: activatorof DsbC/conserved protein [Escherichia coli str. K-12 substr. MG1655](DsbD; C-type cytochrome biogenesis protein CycZ; inner membrane copper tolerance protein; protein-disulfide reductase) (UniProt P36655)   1MAQRIFTLIL LLCSTSVFAG LFDAPGRSQF VPADQAFAFD FQQNQHDLNL TWQIKDGYYL  61YRKQIRITPE HAKIADVQLP QGVWHEDEFY GKSEIYRDRL TLPVTINQAS AGATLTVTYQ 121GCADAGFCYP PETKTVPLSE VVANNAAPQP VSVPQQEQPT AQLPFSALWA LLIGIGIAFT 181PCVLPMYPLI SGIVLGGKQR LSTARALLLT FIYVQGMALT YTALGLVVAA AGLQFQAALQ 241HPYVLIGLAI VFTLLAMSMF GLFTLQLPSS LQTRLTLMSN RQQGGSPGGV FVMGAIAGLI 301CSPCTTAPLS AILLYIAQSG NMWLGGGTLY LYALGMGLPL MLITVFGNRL LPKSGPWMEQ 361VKTAFGFVIL ALPVFLLERV IGDVWGLRLW SALGVAFFGW AFITSLQAKR GWMRIVQIIL 421LAAALVSVRP LQDWAFGATH TAQTQTHLNF TQIKTVDELN QALVEAKGKP VMLDLYADWC 481VACKEFEKYT FSDPQVQKAL ADTVLLQANV TANDAQDVAL LKHLNVLGLP TILFFDGQGQ 541EHPQARVTGF MDAETFSAHL RDRQPSEQ ID NO: 5 NP_415137 thiol:disulfide interchange protein, periplasmic[Escherichia coli str. K-12 substr. MG1655](DsbG; ybdP) (UniProt P77202)   1MLKKILLLAL LPAIAFAEEL PAPVKAIEKQ GITIIKTFDA PGGMKGYLGK YQDMGVTIYL  61TPDGKHAISG YMYNEKGENL SNTLIEKEIY APAGREMWQR MEQSHWLLDG KKDAPVIVYV 121FADPFCPYCK QFWQQARPWV DSGKVQLRTL LVGVIKPESP ATAAAILASK DPAKTWQQYE 181ASGGKLKLNV PANVSTEQMK VLSDNEKLMD DLGANVTPAI YYMSKENTLQ QAVGLPDQKT 241LNIIMGNKSEQ ID NO: 6 NP_417806 FKBP-type peptidyl-prolyl cis-trans isomerase(rotamase) [Escherichia coli str. K-12 substr. MG1655] (FkpA; PPIase)(UniProt P45523)   1MKSLFKVTLL ATTMAVALHA PITFAAEAAK PATAADSKAA FKNDDQKSAY ALGASLGRYM  61ENSLKEQEKL GIKLDKDQLI AGVQDAFADK SKLSDQEIEQ TLQAFEARVK SSAQAKMEKD 121AADNEAKGKE YREKFAKEKG VKTSSTGLVY QVVEAGKGEA PKDSDTVVVN YKGTLIDGKE 181FDNSYTRGEP LSFRLDGVIP GWTEGLKNIK KGGKIKLVIP PELAYGKAGV PGIPPNSTLV 241FDVELLDVKP APKADAKPEA DAKAADSAKKSEQ ID NO: 7 NP_417808 FKBP-type peptidyl prolyl cis-trans isomerase(rotamase) [Escherichia coli str. K-12 substr. MG1655] (SlyD; histidine-rich protein; metallochaperone SlyD; sensitivity to lysis protein D; WHP; PPIase) (UniProt P0A9K9)   1 MKVAKDLVVS LAYQVRTEDG VLVDESPVSA PLDYLHGHGS LISGLETALE GHEVGDKFDV  61 AVGANDAYGQ YDENLVQRVP KDVFMGVDEL QVGMRFLAET DQGPVPVEIT AVEDDHVVVD 121 GNHMLAGQNL KFNVEVVAIR EATEEELAHG HVHGAHDHHH DHDHDGCCGG HGHDHGHEHG 181 GEGCCGGKGN GGCGCHSEQ ID NO: 8 NP_414595 peptidyl-prolyl cis-trans isomerase (PPIase)[Escherichia coli str. K-12 substr. MG1655] (SurA; peptidyl-prolyl cis-trans isomerase SurA; rotamase SurA; survival protein A; PPIase SurA) (UniProt P0ABZ6)   1MKNWKTLLLG IAMIANTSFA APQVVDKVAA VVNNGVVLES DVDGLMQSVK LNAAQARQQL  61PDDATLRHQI MERLIMDQII LQMGQKMGVK ISDEQLDQAI ANIAKQNNMT LDQMRSRLAY 121DGLNYNTYRN QIRKEMIISE VRNNEVRRRI TILPQEVESL AQQVGNQNDA STELNLSHIL 181IPLPENPTSD QVNEAESQAR AIVDQARNGA DFGKLAIAHS ADQQALNGGQ MGWGRIQELP 241GIFAQALSTA KKGDIVGPIR SGVGFHILKV NDLRGESKNI SVTEVHARHI LLKPSPIMTD 301EQARVKLEQI AADIKSGKTT FAAAAKEFSQ DPGSANQGGD LGWATPDIFD PAFRDALTRL 361NKGQMSAPVH SSFGWHLIEL LDTRNVDKTD AAQKDRAYRM LMNRKFSEEA ASWMQEQRAS 421AYVKILSNSEQ ID N0: 9 NP_414720 periplasmic chaperone [Escherichia coli str. K-12substr. MG1655] (Skp; chaperone protein skp; DNA-binding 17 kDa protein;histone-like protein HLP-1; hlpA) (UniProt P0AEU7)   1MKKWLLAAGL GLALATSAQA ADKIAIVNMG SLFQQVAQKT GVSNTLENEF KGRASELQRM  61ETDLQAKMKK LQSMKAGSDR TKLEKDVMAQ RQTFAQKAQA FEQDRARRSN EERGKLVTRI 121QTAVKSVANS QDIDLVVDAN AVAYNSSDVK DITADVLKQV KSEQ ID NO: 10 NP_009887 protein disulfide isomerase PDI1 [Saccharomycescerevisiae S288c] (yPDI; thioredoxin-related glycoprotein 1;TRG1; MFP1)(UniProt P17967)   1MKFSAGAVLS WSSLLLASSV FAQQEAVAPE DSAVVKLATD SFNEYIQSHD LVLAEFFAPW  61CGHCKNMAPE YVKAAETLVE KNITLAQIDC TENQDLCMEH NIPGFPSLKI FKNSDVNNSI 121DYEGPRTAEA IVQFMIKQSQ PAVAVVADLP AYLANETFVT PVIVQSGKID ADFNATFYSM 181ANKHFNDYDF VSAENADDDF KLSIYLPSAM DEPVVYNGKK ADIADADVFE KWLQVEALPY 241FGEIDGSVFA QYVESGLPLG YLFYNDEEEL EEYKPLFTEL AKKNRGLMNF VSIDARKFGR 301HAGNLNMKEQ FPLFAIHDMT EDLKYGLPQL SEEAFDELSD KIVLESKAIE SLVKDFLKGD 361ASPIVKSQEI FENQDSSVFQ LVGKNHDEIV NDPKKDVLVL YYAPWCGHCK RLAPTYQELA 421DTYANATSDV LIAKLDHTEN DVRGVVIEGY PTIVLYPGGK KSESVVYQGS RSLDSLFDFI 481KENGHFDVDG KALYEEAQEK AAEEADADAE LADEEDAIHD ELSEQ ID NO: 11 NP_000909 protein disulfide-isomerase precursor [Homo sapiens] (hPDI; PDI; protein disulfide isomerase-associated 1; DSI; protocollagen hydroxylase; collagen prolyl 4-hydroxylase beta, procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4- hydroxylase), beta polypeptide; prolyl 4-hydroxylase subunit beta;  P4HB; PHDB; PO4DB; PO4HB; PROHB; P4Hbeta; protein disulfideisomerase family A, member 1; PDIA1; protein disulfideisomerase/oxidoreductase; thyroid hormone-binding protein p55; glutathione-insulin transhydrogenase; protein disulfide-isomerase; prolyl 4-hydroxylase subunit beta; cellular thyroid hormone-binding protein; glutathione-insulin transhydrogenase; GIT; ERBA2L) (UniProt P07237)   1MLRRALLCLA VAALVRADAP EEEDHVLVLR KSNFAEALAA HKYLLVEFYA PWCGHCKALA  61PEYAKAAGKL KAEGSEIRLA KVDATEESDL AQQYGVRGYP TIKFFRNGDT ASPKEYTAGR 121EADDIVNWLK KRTGPAATTL PDGAAAESLV ESSEVAVIGF FKDVESDSAK QFLQAAEAID 181DIPFGITSNS DVFSKYQLDK DGVVLFKKFD EGRNNFEGEV TKENLLDFIK HNQLPLVIEF 241TEQTAPKIFG GEIKTHILLF LPKSVSDYDG KLSNFKTAAE SFKGKILFIF IDSDHTDNQR 301ILEFFGLKKE ECPAVRLITL EEEMTKYKPE SEELTAERIT EFCHRFLEGK IKPHLMSQEL 361 PEDWDKQPVK VLVGKNFEDV AFDEKKNVFV EFYAPWCGHC KQLAPIWDKL GETYKDHENI 421VIAKMDSTAN EVEAVKVHSF PTLKFFPASA DRTVIDYNGE RTLDGFKKFL ESGGQDGAGD 481DDDLEDLEEA EEPDMEEDDD QKAVKDELSEQ ID NO: 12 NP_010640 thioredoxin-disulfide reductase TRR1 [Saccharomycescerevisiae S288c] (yTrrl; cytoplasmic thioredoxin reductase) (UniProt P29509)   1MVHNKVTIIG SGPAAHTAAI YLARAEIKPI LYEGMMANGI AAGGQLTTTT EIENFPGFPD  61GLTGSELMDR MREQSTKFGT EIITETVSKV DLSSKPFKLW TEFNEDAEPV TTDAIILATG 121 ASAKRMHLPG EETYWQKGIS ACAVCDGAVP IFRNKPLAVI GGGDSACEEA QFLTKYGSKV 181FMLVRKDHLR ASTIMQKRAE KNEKIEILYN TVALEAKGDG KLLNALRIKN TKKNEETDLP 241VSGLFYAIGH TPATKIVAGQ VDTDEAGYIK TVPGSSLTSV PGFFAAGDVQ DSKYRQAITS 301 AGSGCMAALD AEKYLTSLESEQ ID NO: 13 NP_015234 glutathione-disulfide reductase GLR1 [Saccharomycescerevisiae S288c](yGlrl; glutathione reductase; GR; GRase; LPG17) (UniProt P41921)   1MLSATKQTFR SLQIRTMSTN TKHYDYLVIG GGSGGVASAR RAASYGAKTL LVEAKALGGT  61CVNVGCVPKK VMWYASDLAT RVSHANEYGL YQNLPLDKEH LTFNWPEFKQ KRDAYVHRLN 121GIYQKNLEKE KVDVVFGWAR FNKDGNVEVQ KRDNTTEVYS ANHILVATGG KAIFPENIPG 181FELGTDSDGF FRLEEQPKKV VVVGAGYIGI ELAGVFHGLG SETHLVIRGE TVLRKFDECI 241QNTITDHYVK EGINVHKLSK IVKVEKNVET DKLKIHMNDS KSIDDVDELI WTIGRKSHLG 301MGSENVGIKL NSHDQIIADE YQNTNVPNIY SLGDVVGKVE LTPVAIAAGR KLSNRLFGPE 361 KFRNDKLDYE NVPSVIFSHP EAGSIGISEK EAIEKYGKEN IKVYNSKFTA MYYAMLSEKS 421PTRYKIVCAG PNEKVVGLHI VGDSSAEILQ GFGVAIKMGA TKADFDNCVA IHPTSAEELV 481TMRSEQ ID NO: 14 NP_414970 peptidyl-prolyl cis/trans isomerase (trigger factor) [Escherichia coli str. K-12 substr. MG1655](tig; TF; ECK0430; JW0426; PPIase) (UniProt P0A850)   1MQVSVETTQG LGRRVTITIA ADSIETAVKS ELVNVAKKVR IDGFRKGKVP MNIVAQRYGA  61SVRQDVLGDL MSRNFIDAII KEKINPAGAP TYVPGEYKLG EDFTYSVEFE VYPEVELQGL 121EAIEVEKPIV EVTDADVDGM LDTLRKQQAT WKEKDGAVEA EDRVTIDFTG SVDGEEFEGG 181KASDFVLAMG QGRMIPGFED GIKGHKAGEE FTIDVTFPEE YHAENLKGKA AKFAINLKKV 241EERELPELTA EFIKRFGVED GSVEGLRAEV RKNMERELKS AIRNRVKSQA IEGLVKANDI 301DVPAALIDSE IDVLRRQAAQ RFGGNEKQAL ELPRELFEEQ AKRRVVVGLL LGEVIRTNEL 361KADEERVKGL IEEMASAYED PKEVIEFYSK NKELMDNMRN VALEEQAVEA VLAKAKVTEK 421ETTFNELMNQ QASEQ ID NO: 15 NP_000933 peptidyl-prolyl cis-trans isomerase B precursor [Homo sapiens] (hPPIB; PPIase B; PPIB; rotamase B; cyclophilin B; cyclophilin-like protein; S-cyclophilin; SCYLP; CYP-S1; CYPB) (UniProt P23284)   1MLRLSERNMK VLLAAALIAG SVFFLLLPGP SAADEKKKGP KVTVKVYFDL RIGDEDVGRV  61IFGLFGKTVP KTVDNFVALA TGEKGFGYKN SKFHRVIKDF MIQGGDFTRG DGTGGKSIYG 121 ERFPDENFKL KHYGPGWVSM ANAGKDTNGS QFFITTVKTA WLDGKHVVFG KVLEGMEVVR 181KVESTKTDSR DKPLKDVIIA DCGKIEVEKP FAIAKESEQ ID NO: 16 NP_010439 peptidylprolyl isomerase CPR1 [Saccharomycescerevisiae S288c](Cprl, peptidyl-prolyl cis-trans isomerase, cyclophilin,CPH1, CYP1, cyclosporin A-binding protein, rotamase, PPIase, PPI-II) (UniProt P14832)   1MSQVYFDVEA DGQPIGRVVF KLYNDIVPKT AENFRALCTG EKGFGYAGSP FHRVIPDFML  61QGGDFTAGNG TGGKSIYGGK FPDENFKKHH DRPGLLSMAN AGPNTNGSQF FITTVPCPWL 121DGKHVVFGEV VDGYDIVKKV ESLGSPSGAT KARIVVAKSG ELSEQ ID NO: 17 NP_013317 peptidylprolyl isomerase CPR6 [Saccharomycescerevisiae S288c] (Cpr6, cyclophilin, CYP40, rotamase CPR6, PPIase CPR6)(UniProt P53691)   1MTRPKTFFDI SIGGKPQGRI VFELYNDIVP KTAENFLKLC EGNAGMAKTK PDVPLSYKGS  61 IFHRVIKDFM CQFGDFTNFN GTGGESIYDE KFEDENFTVK HDKPFLLSMA NAGPNTNGSQ 121AFITCVPTPH LDGKHVVFGE VIQGKRIVRL IENQQCDQEN NKPLRDVKID DCGVLPDDYQ 181VPENAEATPT DEYGDNYEDV LKQDEKVDLK NFDTVLKAIE TVKNIGTEQF KKQNYSVALE 241KYVKCDKFLK EYFPEDLEKE QIEKINQLKV SIPLNIAICA LKLKDYKQVL VASSEVLYAE 301AADEKAKAKA LYRRGLAYYH VNDTDMALND LEMATTFQPN DAAILKAIHN TKLKRKQQNE 361KAKKSLSKMF SSEQ ID NO: 18 NP_014264 peptidylprolyl isomerase FPR1 [Saccharomycescerevisiae S288c] (Fprl, FK506-binding protein 1, FKBP, FKB1, rapamycin-binding protein, RBP1, PPIase) (UniProt P20081)   1MSEVIEGNVK IDRISPGDGA TFPKTGDLVT IHYTGTLENG QKFDSSVDRG SPFQCNIGVG  61QVIKGWDVGI PKLSVGEKAR LTIPGPYAYG PRGFPGLIPP NSTLVFDVEL LKVNSEQ ID NO: 19 NP_057390 dnaJ homolog subfamily B member 11 precursor [Homosapiens] (hERdj3; DnaJ (Hsp40) homolog, subfamily B, member 11; ER-associated DNAJ; ER-associated Hsp40 co-chaperone; ER-associated dnaJ protein 3; ERdj3; ERj3p; EDJ; ERJ3; ERj3; HEDJ; human DnaJ protein 9; DnaJ protein homolog 9HDJ9; DJ9; Dj-9; hDj-9; PWP1-interacting protein 4; APOBEC1-binding protein 2; ABBP-2; ABBP2; DNAJB11; PRO1080; UNQ537) (UniProt Q9UBS4)   1MAPQNLSTFC LLLLYLIGAV IAGRDFYKIL GVPRSASIKD IKKAYRKLAL QLHPDRNPDD  61PQAQEKFQDL GAAYEVLSDS EKRKQYDTYG EEGLKDGHQS SHGDIFSHFF GDFGFMFGGT 121PRQQDRNIPR GSDIIVDLEV TLEEVYAGNF VEVVRNKPVA RQAPGKRKCN CRQEMRTTQL 181 GPGRFQMTQE VVCDECPNVK LVNEERTLEV EIEPGVRDGM EYPFIGEGEP HVDGEPGDLR 241FRIKVVKHPI FERRGDDLYT NVTISLVESL VGFEMDITHL DGHKVHISRD KITRPGAKLW 301KKGEGLPNFD NNNIKGSLII TFDVDFPKEQ LTEEAREGIK QLLKQGSVQK VYNGLQGYSEQ ID NO: 20 NP_005338 78 kDa glucose-regulated protein precursor [Homosapiens](BiP; endoplasmic reticulum lumenal Ca(2+)-binding protein grp78;GRP-78; heat shock 7C kDa protein 5; HSPA5; immunoglobulin heavy chain-binding protein; MIF2) (UniProt P11021)   1MKLSLVAAML LLLSAARAEE EDKKEDVGTV VGIDLGTTYS CVGVFKNGRV EIIANDQGNR  61ITPSYVAFTP EGERLIGDAA KNQLTSNPEN TVFDAKRLIG RTWNDPSVQQ DIKFLPFKVV 121EKKTKPYIQV DIGGGQTKTF APEEISAMVL TKMKETAEAY LGKKVTHAVV TVPAYFNDAQ 181RQATKDAGTI AGLNVMRIIN EPTAAAIAYG LDKREGEKNI LVFDLGGGTF DVSLLTIDNG 241VFEVVATNGD THLGGEDFDQ RVMEHFIKLY KKKTGKDVRK DNRAVQKLRR EVEKAKRALS 301SQHQARIEIE SFYEGEDFSE TLTRAKFEEL NMDLFRSTMK PVQKVLEDSD LKKSDIDEIV 361LVGGSTRIPK IQQLVKEFFN GKEPSRGINP DEAVAYGAAV QAGVLSGDQD TGDLVLLDVC 421PLTLGIETVG GVMTKLIPRN TVVPTKKSQI FSTASDNQPT VTIKVYEGER PLTKDNHLLG 481TFDLTGIPPA PRGVPQIEVT FEIDVNGILR VTAEDKGTGN KNKITITNDQ NRLTPEEIER 541MVNDAEKFAE EDKKLKERID TRNELESYAY SLKNQIGDKE KLGGKLSSED KETMEKAVEE 601KIEWLESHQD ADIEDFKAKK KELEEIVQPI ISKLYGSAGP PPTGEEDTAE KDELSEQ ID NO: 21 NP_013911 Hsp90 family chaperone HSC82 [Saccharomycescerevisiae S288c](yHsc82; HSC82; ATP-dependent molecular chaperone HSC82; 82 kDa heat shock cognate protein; heat shock protein Hsp90 constitutiveisoform; HSP90; cytoplasmic chaperone of the Hsp90 family) (UniProt P15108)  1 MAGETFEFQA EITQLMSLII NTVYSNKEIF LRELISNASD ALDKIRYQAL SDPKQLETEP 61 DLFIRITPKP EEKVLEIRDS GIGMTKAELI NNLGTIAKSG TKAFMEALSA GADVSMIGQF121 GVGFYSLFLV ADRVQVISKN NEDEQYIWES NAGGSFTVTL DEVNERIGRG TVLRLFLKDD 181 QLEYLEEKRI KEVIKRHSEF VAYPIQLLVT KEVEKEVPIP EEEKKDEEKK DEDDKKPKLE241 EVDEEEEEKK PKTKKVKEEV QELEELNKTK PLWTRNPSDI TQEEYNAFYK SISNDWEDPL301 YVKHFSVEGQ LEFRAILFIP KRAPFDLFES KKKKNNIKLY VRRVFITDEA EDLIPEWLSF361 VKGVVDSEDL PLNLSREMLQ QNKIMKVIRK NIVKKLIEAF NEIAEDSEQF DKFYSAFAKN421 IKLGVHEDTQ NRAALAKLLR YNSTKSVDEL TSLTDYVTRM PEHQKNIYYI TGESLKAVEK481 SPFLDALKAK NFEVLFLTDP IDEYAFTQLK EFEGKTLVDI TKDFELEETD EEKAEREKEI541 KEYEPLTKAL KDILGDQVEK VVVSYKLLDA PAAIRTGQFG WSANMERIMK AQALRDSSMS601 SYMSSKKTFE ISPKSPIIKE LKKRVDEGGA QDKTVKDLTN LLFETALLTS GFSLEEPTSF661 ASRINRLISL GLNIDEDEET ETAPEASTEA PVEEVPADTE MEEVDSEQ ID NO: 22 NP_418142 heat shock chaperone [Escherichia coli str. K-12substr. MG1655] (IbpA; small heat shock protein IbpA; 16 kDa heat shockprotein A; hs1T; htpN; ECK3679; JW3664) (UniProt P00054)   1 MRNFDLSPLY RSAIGFDRLF NHLENNQSQS NGGYPPYNVE LVDENHYRIA IAVAGFAESE  61LEITAQDNLL VVKGAHADEQ KERTYLYQGI AERNFERKFQ LAENIHVRGA NLVNGLLYID 121LERVIPEAKK PRRIEINSEQ ID NO: 23 NP_418141 heat shock chaperone [Escherichia coli str. K-12substr. MG1655] (IbpB; small heat shock protein IbpB; 16 kDa heat shockprotein B; hs1S; htpE; ECK3678; JW3663) (UniProt P00058)   1MRNFDLSPLM RQWIGFDKLA NALQNAGESQ SFPPYNIEKS DDNHYRITLA LAGFRQEDLE  61 IQLEGTRLSV KGTPEQPKEE KKWLHQGLMN QPFSLSFTLA ENMEVSGATF VNGLLHIDLI 121RNEPEPIAAQ RIAISERPAL NS

1. A method of improving the expression levels of biologically activeproteins in a bacterial cell free synthesis system comprising the stepsof: i) preparing a bacterial extract having an active oxidativephosphorylation system and comprising biologically functioning tRNA,amino acids and ribosomes necessary for cell free protein synthesis,wherein the bacteria from which the extract is prepared expresses anexogenous protein chaperone at a concentration of at least about 1gm/liter of extract; ii) combining the bacterial extract with a nucleicacid encoding a protein of interest to yield a bacterial cell freesynthesis system; and, iii) incubating the bacterial cell free synthesissystem under conditions permitting the expression of the protein ofinterest to a concentration of at least about 100 mg/L. 2-10. (canceled)11. A bacterial cell free synthesis system for expressing biologicallyactive proteins comprising: i) a cell free extract of bacteria having anactive oxidative phosphorylation system, containing biologicallyfunctioning tRNA, amino acids and ribosomes necessary for cell freeprotein synthesis and wherein an exogenous protein chaperone wasexpressed in the bacteria at a level of at least 1 gm/liter of extract;and ii) a nucleic acid encoding a protein of interest, where saidbacterial cell free synthesis system expresses a protein of interest toa concentration of at least about 100 mg/L.
 12. The system of claim 11,wherein the protein chaperone is selected from the group consisting ofdisulfide isomerases and prolyl isomerases.
 13. The system of claim 11,wherein the bacteria from which the extract is prepared wasco-transformed with genes encoding disulfide isomerases and prolylisomerases.
 14. The system of claim 11, wherein the exogenous proteinchaperone is selected from the group consisting of DsbC, FkpA, and SlyD.15. The system of claim 11, wherein the bacteria is Escherichia coli.16. The system of claim 11, wherein the bacteria from which the extractis prepared expresses the exogenous protein chaperone from a geneoperably linked to a constitutive promoter.
 17. The system of claim 11,wherein the extract is an S30 extract of E. coli.
 18. A method ofexpressing properly folded, biologically active proteins in a bacterialcell free synthesis system comprising the steps of: i) preparing abacterial extract comprising biologically functioning tRNA, amino acids,ribosomes necessary for cell free protein synthesis, a protein disulfideisomerase and a peptidyl-prolyl cis-trans isomerase, wherein the proteindisulfide isomerase and the peptidyl-prolyl cis-trans isomerase arepresent at a concentration sufficient to improve the expression ofproperly folded biologically active proteins; ii) combining thebacterial extract with a nucleic acid encoding a protein of interest;and iii) incubating the bacterial extract with the nucleic acid underconditions permitting the expression and proper folding of the proteinof interest. 19-27. (canceled)
 28. A bacterial cell free synthesissystem for expressing biologically active proteins in a comprising: i) acell free extract of bacteria having an active oxidative phosphorylationsystem, containing biologically functioning tRNA, amino acids andribosomes necessary for cell free protein synthesis and furtherincluding a protein disulfide isomerase and a peptidyl-prolyl cis-transisomerase, wherein the protein disulfide isomerase and thepeptidyl-prolyl cis/trans isomerase are present at a concentrationsufficient to improve the expression of properly folded biologicallyactive proteins; and ii) a nucleic acid encoding a protein of interest,wherein said bacterial cell free synthesis system expresses a protein ofinterest to a concentration of at least about 100 mg/L.
 29. The systemof claim 28, wherein the total concentration of a protein disulfideisomerase and a peptidyl-prolyl cis-trans isomerase are present in aconcentration of at least about 1 gm/liter in the extract.
 30. Thesystem of claim 28, wherein the total concentration of a proteindisulfide isomerase and a peptidyl-prolyl cis-trans isomerase arepresent in a concentration of between 1 gm/liter and 14 gm/liter in theextract.
 31. The system of claim 28, wherein the expression of theprotein of interest is improved to a concentration above thatconcentration where one but not both of the protein disulfide isomeraseand the peptidyl-prolyl cis-trans isomerase are present, and theincubation conditions are otherwise the same.
 32. The system of claim28, wherein the protein disulfide isomerase is selected from the groupconsisting of DsbA, DsbB, DsbC, and DsbD, and the peptidyl-prolylcis-trans isomerase is selected from the group consisting of FkpA andSlyD.
 33. The system of claim 28, wherein the bacteria from which theextract is prepared expresses at least one of the exogenous proteinchaperones from a gene operably linked to a constitutive promoter. 34.The system of claim 28, wherein the bacteria is Escherichia coli. 35.The system of claim 28, wherein the protein of interest has at least onedisulfide bond in its biologically active conformation.
 36. The systemof claim 28, wherein the protein of interest has at least two prolineresidues.
 37. The system of claim 28, wherein the protein of interest isan antibody or antibody fragment.